Global gene regulators (GGR) as vaccine candidates against paratuberculosis

ABSTRACT

Described herein is a  mycobacterium  mutant, comprising at least one mutation in at least one gene sequence encoding global gene regulators (GGRs) selected from the group consisting of sigH, sigL, sigE, ECF-1, and mixtures thereof, wherein the GGR gene is at least partially inactivated. Described herein also is a vaccine based on the mutant and a method of differentiating between subjects that have been infected with  mycobacterium  and subjects that have not been infected with  mycobacterium  or have been vaccinated with a  mycobacterium  vaccine.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims priority to U.S. Ser. No. 61/777,907, filed Mar.12, 2013, which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 14-CRHF-0-6055awarded by the USDA/NIFA. The government has certain rights in theinvention.

BACKGROUND

Mycobacterium avium subspecies paratuberculosis (a.k.a. M.paratuberculosis) is the etiological agent of Johne's disease, a chronicenteritis of domestic and wild animals, especially ruminants. Johne'sdisease (JD) has been reported on every continent (1-3), and isconsidered one of the greatest causes of economic hardship to theruminant industries (4). More than two thirds of the U.S. dairy herdsare infected with JD (5) and this wide distribution of the disease,reduced milk production and premature culling of infected animalstogether causes severe economic losses estimated to be over $200 milliona year for the dairy industry (6,7).

Majority of the M. paratuberculosis infection occurs through fecal-oralroute and the mycobacteria are endocytosed by enterocytes and M cells inthe Peyer's patches of the ileum (8,9). After subsequent internalizationby subepithelial and intraepithelial macrophages, M. paratuberculosis isable to survive and persist within the cells (10) using mechanisms thatare not completely understood. Several studies examined gene expressionpatterns and host defense mechanisms of bovine macrophages fromnaturally infected cows (11), peripheral blood mononuclear cells (PBMC)(12) or monocytes-derived macrophages (MDMs) (13,14) following infectionwith M. paratuberculosis. Alternatively, our group characterized thegeneral and specific stress responses of M. paratuberculosis undervarious in vitro conditions as well as the transcriptomes of M.paratuberculosis in fecal samples from diseased cows (15).

Survival of M. paratuberculosis in environmental samples (16),macrophages (17) and animal models (18,19) is well-documented, however,the genetic basis for this survival remains unknown. Reports employing alarge-scale screening of M. paratuberculosis mutants in relevant animalmodels (20,21) provided some insights into virulence of this organismwith the identification of novel virulence factors associated withbiofilm formation (22) and epithelial cell invasion (23). Recently, Zhu,et al. analyzed intracellular M. paratuberculosis gene expressionpatterns in bovine MDMs using SCOTS (selective capture of transcribedsequences), identifying similar patterns of responses to oxidativestress, metabolic activity, and cell survival among M. paratuberculosiswith distinct host origins (24). The same group further analyzed theexpression profiles of M. paratuberculosis isolated from naturallyinfected bovine tissues, identifying tissue-specific pathways (25).However, no comprehensive study has been conducted to clarify therelationship between M. paratuberculosis gene expression and specifichost microenvironments following macrophage infection.

The current vaccine has a limited use to farmers in some regions (e.g. afew European countries) because of its inability to reduce M. apshedding in feces of infected animals, the main source for spreading JD.There is only one vaccine (MYCOPAR, Boehringer Ingelheim) approved forlimited use in the USA. This vaccine causes significant granulomaformation at the site of inoculation 21, which persists throughout theanimal's life, increasing the possibility for tissue condemnation at theslaughterhouse.

Despite the ability of this vaccine to induce cell mediated immunity inanimals¹⁷, shedding of M. ap from vaccinated animals continues to causea problem for transmitting the disease to naïve animals (5, 22). Insheep, some animals both shed M. ap and died from multi-bacillary formof JD despite being vaccinated (23). In a long-term study of the effectof killed vaccine on dairy herds to reduce the transmission of thedisease, no significant difference in prevalence was found betweenvaccinated and non-vaccinated herds (22). In another study of commercialJD vaccines, cross reactivity to bovine tuberculosis was prominent,further hampering efforts for controlling tuberculosis in farm animals(24,25). More efforts are needed to better understand the pathogenesisof JD and to plan an effective control strategy.

The present invention starts with a goal to gain insights into how M.paratuberculosis respond to the intracellular microenvironments ofmacrophages, the primary site for mycobacterial persistence within thehost, using targeted mutagenesis and an array of transcriptome analyses.In this study, the inventors took advantage of analytical microscopy todefine the phagosome environment of M. paratuberculosis-containingmacrophages in association with the expression profile of mycobacterialbacilli using DNA microarrays. The analysis suggested key changes in themetabolic pathways of M. paratuberculosis once the bacteria encounteractive macrophages and the activation of various alternative sigmafactors (Global Gene Regulators, GGRs) that could help M.paratuberculosis survive the hostile intracellular environment ofmacrophages. One such alternative GGR, sigH, has been shown tocontribute to the resistance encountered during variable environmentalstress conditions, such as temperature and oxidative stress in M.tuberculosis (26,27). However, the basis of transcriptional regulationof sigH remains elusive in M. paratuberculosis.

The inventors therefore sought to define the gene regulatory networkunder control of GGRs, for example, sigH, sigL, sigE and ECF-1 in M.paratuberculosis. Accordingly, they confirmed a role for these key GGRsactivated inside macrophages in defending M. paratuberculosis againstthiol-specific oxidative stress and characterized the effect of theseGGRs on global transcriptome in M. paratuberculosis. Based on theresults, the inventor envisions that the GGR mutants could play animportant role in designing effective vaccines against mycobacterialinfections.

BRIEF SUMMARY OF THE INVENTION

In its first aspect, the present invention relates to a mycobacteriummutant, comprising at least one mutation in at least one gene sequenceencoding global gene regulators (GGRs) selected from the groupconsisting of sigH, sigL, sigE, ECF-1, and mixtures thereof, wherein theGGR gene is at least partially inactivated.

In some embodiments, the mycobacterium is selected from the groupconsisting of Mycobacterium avium subspecies paratuberculosis (M. ap),Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium avium subsp. avium (M. avium), and mixturesthereof. Preferably, the mycobacterium is M. ap, M. bovis, or M.tuberculosis.

In some embodiments, the GGR is M. ap sigH (SEQ ID NO:1) or a sequencesubstantially identical to SEQ ID NO:1. In some embodiments, the GGR isM. ap sigL (SEQ ID NO:2) or a sequence substantially identical to SEQ IDNO:2. In some embodiments, the GGR is M. ap sigE (SEQ ID NO:3) or asequence substantially identical to SEQ ID NO:3. In some embodiments,the GGR is M. ap ECF-1 (SEQ ID NO:4) or a sequence substantiallyidentical to SEQ ID NO:4. In other embodiments, the GGR is M. ap sigB(SEQ ID NO:5) or a sequence substantially identical to SEQ ID NO:5.

In some embodiments, the inactivation of the GGR genes is achieved by atleast partial deletion of the gene sequence encoding GGRs. Preferably,the inactivation is achieved by a complete deletion of the gene sequenceencoding GGRs.

In some embodiments, the inactivation of the GGR genes is achieved by atleast partial transposon insertion of the gene sequence encoding GGRs.Preferably, the inactivation is achieved by a complete transposoninsertion of the gene sequence encoding GGRs.

In some embodiments, the inactivation of the GGR genes is achieved by atleast partial anti-sense construct of the gene sequence encoding GGRs.Preferably, the inactivation is achieved by a complete anti-senseconstruct of the gene sequence encoding GGRs.

In its second aspect, the present invention relates to an isolatedmycobacterial organism comprising a mycobacterium mutant describedherein.

In its third aspect, the present invention relates a mycobacteriumvaccine comprising a mycobacterium mutant described herein. In someembodiments, the vaccine may also comprise the mycobacterium organismdescribed above.

In some embodiments, the vaccine is physically inactivated. Preferably,the vaccine is heat inactivated.

In some embodiments, the vaccine is chemically inactivated. Preferably,the vaccine is formaldehyde inactivated.

In other embodiments, the vaccine is a live attenuated vaccine.

In its fourth aspect, the present invention relates to a method ofdifferentiating between subjects that have been infected withmycobacterium and subjects that have not been infected withmycobacterium or have been vaccinated with a mycobacterium vaccine.

In one embodiment, the method comprises the steps of identifying asequence that is specific to a mycobacterium mutant and is not found inthe wild-type mycobacterium strain; and detecting the presence of thesequence in the subject. In another embodiment, the method comprises thesteps of identifying a sequence that is specific to a wild-typemycobacterium strain and is not found in the mycobacterium mutant; anddetecting the presence or the quantity of the sequence in the subject.

In a preferred embodiment, the mycobacterium mutant is a mutantdescribed herein. In another preferred embodiment, the detection is madeby using probes or primers complementary to the mycobacterium mutant inan amplification reaction. More preferably, the amplification is theloop-mediated isothermal amplification (LAMP).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Temporal expression of M. avium subsp. paratuberculosis sigmafactors within macrophages using DNA microarrays. A selected list of theM. avium subsp. paratuberculosis sigma factor genes are shown undernaive (A) and activated (B) macrophages. Note that sigH and other ECF-1(ECF-1 on the chart for clarity) were upregulated immediately afterinfection, followed by expression of sigE and sigB.

FIG. 2. Survival of M. avium subsp. paratuberculosis strains in bovinemacrophages. Naive (A) and IFN-γ-pretreated (B) MDM cells were infectedwith ΔsigH mutant and wild-type M. avium subsp. paratuberculosisstrains. Cells were lysed at 1, 4, and 8 days postinfection, and numbersof viable bacilli were determined by CFU plating. The survival levels at4 and 8 days were relative to the viable counts of bacterial strains atday 1. Survival data represent the average of macrophage infectionscollected from three different donor animals with significance levels inStudent's t test (*, P<0.05). Error bars represent the standarddeviations.

FIG. 3. Virulence of M. avium subsp. paratuberculosis K10 and the ΔsigHmutant in mice. Mice groups (n=15) were inoculated with ˜2×10⁸ CFU/mouseof M. avium subsp. paratuberculosis wild-type strain or the ΔsigH mutantvia intraperitoneal injection. Intestines (A) and spleens (B) werecollected at 3, 6, and 12 wpi (n=5 mice/group/time point) and culturedfor bacterial counts. Colony counts for each group are represented byscattered plots accompanied with a median line. Organs with significantdifference in bacterial load were denoted with * for P values of <0.05or ** for P values of <0.01). (C) Pathology of spleen collected frommice infected with M. avium subsp. paratuberculosis K10 (a) and itsisogenic ΔsigH mutant (b). H&E-stained sections with 100× magnification(scale bar=200 μm) are shown. Inset images (1,000× magnification, scalebar=10 μm) show the M. avium subsp. paratuberculosis bacilli in purplecolor (arrows). WP, white pulp; RP, red pulp.

FIG. 4. Construction of M. avium subsp. paratuberculosis ΔsigL usingwild-type M. avium subsp. paratuberculosis strain. (A) A Physical mapdisplaying the deletion of sigL (MAP4201) gene with homologousrecombination via pYUB854 cosmid shuttle cloning vector, which resultedin the deletion of ˜750 bp coding region and the insertion of a ˜2 kbregion encoding a hygromycin resistance cassette. (B) The M. aviumsubsp. paratuberculosis ΔsigL mutant was confirmed with PCR and sequenceverification using genomic DNA from the wild-type and the mutantstrains. Primer pairs were designed for the sigL region, hygromycinresistance gene (hyg^(r)), or the recombinant region after allelicexchange. A 1.5% agarose gel showed amplicons from the sigL region onlywhen wild-type genomic DNA was used (lane 1), whereas hyg^(r) wasamplified only from the M. avium subsp. paratuberculosis ΔsigL mutantgenomic DNA (lane 4). Lane 6 showed amplicon from the recombinant regiononly when M. avium subsp. paratuberculosis ΔsigL mutant genomic DNA wasused. (C) The polarity of the M. avium subsp. paratuberculosis ΔsigLknockout mutant was assessed using reverse-transcriptase PCR analysis tocheck for transcription of its neighboring genes. In the wild-type(left) and complemented (right) strains, positive bands show that map′,sigL and the downstream gene MAP4202 are both encoded in the genome andtranscribed (amplified from cDNA), with no amplification from RNA usedas a negative control. In the M. avium subsp. paratuberculosis ΔsigLmutant (middle), the sigL coding region is absent in the genome or ascDNA, but transcripts for the neighboring genes are present.

FIG. 5. Analysis of immune responses in immunized mice before challenge.(A) Scheme illustrating the immunization study. C57BL/6 mice received atotal of 2 doses, each containing ˜2×10⁶ CFU M. avium subsp.paratuberculosis ΔsigL by s.c injection. Mock group received PBS buffer.Following vaccination, both groups of mice were challenged withwild-type M. avium subsp. paratuberculosis strain as described above.After 6 weeks post-immunization (6 PWI; week 0 in the scheme) or 6 weekspost-challenge (WPC), mice (N=4-6) from each group were sacrificed foranalysis of immune response. (B) Splenocytes (6 PWI) were isolated andre-stimulated in vitro with Johnin PPD to measure IFN-γ levels fromculture supernatant by ELISA after 48 h. (C) M. avium subsp.paratuberculosis specific antibody (anti-PPDj antibodies) in the mousesera (6 PWI) was detected by ELISA (OD 450 nm) using Horseradishperoxidase conjugated rabbit anti-mouse antibody. *p<0.05.

FIG. 6. Protective efficacy of immunization. At 6 weeks followingvaccination, mice received a challenge dose containing ˜7×10⁸ CFUwild-type M. avium subsp. paratuberculosis strain i.p (see FIG. 5A).Following challenge, mice groups (N=4) were sacrificed at 6 and 12 weeksand bacterial burden was analyzed in spleen (A), liver (B), andintestine (C) organs. Horizontal lines indicate median value.Statistical analyses were done using student's t test and Mann-Whitneytest to evaluate differences in bacterial organ load among mice groupsvaccinated with the PBS (mock) or M. avium subsp. paratuberculosis ΔsigLmutant. (D) Secretion of IFN-γ production (6 WPC) from the cellsupernatant was measured by ELISA. The histograms show mean values witherror bars representing the standard deviation. *p<0.05, **p<0.01.

FIG. 7. Pathological analysis of mice organs following vaccination.Photographs shows haematoxylin and eosin staining liver (A-B) andintestine (C-D) section (100× magnification, scale bar=200 μm) from mockand M. avium subsp. paratuberculosis ΔsigL vaccinated animals followingchallenge with wild-type M. avium subsp. paratuberculosis strain at 6WPC and 12 WPC. Ziehl-Neelsen staining of both liver and intestinedisplayed higher acid-fast bacilli (inset images; 1000× magnification,scale bar=10 μm) in the mock vaccinated animals compared to the onesthat received M. avium subsp. paratuberculosis ΔsigL vaccination.

FIG. 8. Virulence of M. ap mutants in BALB/c mice (n=15/group) followingIP. A) Spleen (circle) and B) Liver (square) were collected at 3, 6, and12 WPI. Colonization levels were compared to M. ap K10 (Continuedline). * denotes significant difference between groups in Student'st-test (p<0.05).

FIG. 9. Pathology of spleen collected from mice infected with wild typeM. ap (a), and its isogenic mutant ΔsigH (b). H&E stained sections with100× (Bar=200 μm) are shown. Inset images (1000×, Bar=10 μm). WP, whitepulp; RP, red pulp.

FIG. 10. Vaccine potential of M. ap σ factors deleted mutants. Recoveryof virulent M. ap (cfu/g tissue) from A) Intestine and B) Liver of miceimmunized with mock (PBS), ΔsigL or ΔsigH at 6 and 12 weeks followingchallenge. Colony forming unit (cfu) counts are shown as a scatter plotwhere the bar represents the median. *p<0.05.

DESCRIPTION OF THE INVENTION

The present invention provides mycobacterium mutants and a vaccine basedon the mutants for successfully generating immune response to theinfection by Mycobacteria, and in particular for generating immuneresponse to infection caused by Mycobacterium avium subspeciesparatuberculosis (M. ap). The present invention is based on theidentification of the gene regulatory network under control of globalgene regulators (GGRs) (e.g. sigma factors, transcriptional regulators).It is found that some of key GGRs can be genetically mutated to beactivated inside macrophages in defending M. paratuberculosis againstthiol-specific oxidative stress and characterized the effect of GGRs onglobal transcriptome in M. paratuberculosis. It is envisioned that amutant with the deletion, inactivation or reduction of GGRs gene couldprovide strains capable of replication in hosts to generate enoughprotective immunity.

DEFINITIONS

A “mycobacterium” as used herein, refers to micro-organisms of the genusMycobacterium (sometimes abbreviated as M. herein), from the family ofMycobacteriaceae. The particularly interested mycobacteria for thepurpose of the present invention include members of Mycobacterium aviumsubspecies paratuberculosis (M. ap), mycobacterium bovis (M. bovis),Mycobacterium avium subsp. avium (M. avium), M. bovis BCG (the strainmost often used for vaccination purposes), mycobacterium tuberculosis(M. tuberculosis), which includes the species M. tuberculosis (the majorcause of human tuberculosis), M. africanum, M. microti, M. canetti, andM. pinnipedii.

A “global gene regulator, GGR” refers to any protein needed forinitiation of RNA synthesis, for example, “sigma factors” and“transcriptional regulators”. They are bacterial transcriptioninitiation factors that enable specific binding of RNA polymerase togene promoters. RNA polymerase holoenzyme complex consists of core RNApolymerase and a GGR executes transcription of a DNA template strand.The specific GGR used to initiate transcription of a given gene willvary, depending on the gene and on the environmental signals needed toinitiate transcription of that gene.

A “mutation” as used herein refers to a mutation in the genetic material(typically DNA), in particular a non-naturally occurring mutation,obtained via genetic engineering techniques. Mutations may includeinsertions, deletions, anti-sense constructs, substitutions (e.g.,transitions, transversion), transpositions, inversions and combinationsthereof. Mutations also include mutations upstream of the start codon,such as in the promoter region, particularly mutations within 30nucleotides upstream of the start codon, as long as these mutationsaffect the levels and/or function of the gene product. Mutations mayinvolve only a single nucleotide (e.g., a point mutation or a singlenucleotide polymorphism) or multiple nucleotides.

In some embodiments, the mutation is a partial or complete deletion ofthe gene. In some embodiments, the mutation is introduced by insertingheterologous sequences into the gene of interest. In other embodiments,the mutation is introduced by replacing a portion of the wild-type geneor allele, or a majority of the wild-type gene or allele, with aheterologous sequence, or an engineered (e.g., manually altered,disrupted, or changed), non-functional, copy of the wild-type sequence.In some embodiments, the mutation is introduced by a partial or completeanti-sense construct, which refers to a DNA sequence complementary to aparticular gene. When such construct is introduced in the genome, itwill be transcribed in RNA and will be paired with the RNA of the geneit is complementary to. This complex of two RNA (double stranded RNA)will then be degraded so that no protein of the gene will be expressed.

Preferably, the mutation is done by deleting at least about 10%, atleast about 20%, at least about 30%, at least about 50%, at least about70% or at least 90% of the wild-type gene sequence. In otherembodiments, the mutation is done by deleting the full wild-typesequence. In some other embodiments, the mutation may even include adeletion of more than 100% of the wild-type gene sequence (e.g., thefull wild-type gene sequence may be deleted but also include one or moreheterologous and/or non-functional nucleic acid sequences attachedthereto or inserted therein).

Mutations could be silent, that is, no phenotypic effect of the mutationis detected. On the other hand, mutations may also cause a phenotypicchange. For example, the expression level of the encoded product isaltered, or the encoded product itself is altered. It is also called asthe gene with the genetically engineered mutation encodes a gene productthat has a reduced (knock-down) or absent (knock-out) functionality.This may be either because there is none or less of the gene productpresent than in the corresponding wild type strain, and/or because thegene product is not or only partially functional.

Thus, the mutations may influence expression levels of the gene product,stability of the gene product, encode defunct or nonsense gene products(e.g. by insertion of a stop codon), encode a different gene product, orany combination of these. For example, a mutation may result in adisrupted gene with decreased levels of expression of a gene product(e.g., protein or RNA) as compared to the wild-type strain (e.g., M.ap). A mutation may also result in an expressed protein with activitythat is lower as compared to the activity of the expressed protein fromthe wild-type strain (e.g., M. ap). In some embodiments, the activity ofthe protein is reduced by 10%, 30%, 50%, 70%, 90% or more.

In specific embodiments of the present invention, mutations areconducted in at least one gene sequence encoding global gene regulators(GGRs). Such mutations result in at least partial inactivation of thefunction of the GGR genes. The term “function” as used herein isintended to mean the function of GGR gene sequences directly and/orindirectly attributable to the expression of the GGR genes. The“function” also refers to the function and/or activity of the proteinsencoded by GGR genes as to their indirectly and/or directly attributionto infectious and/or inflammasome activation of mycobacteria.

The term “at least partially inactivated” as used herein, is meant toindicate a loss, interruption, or alteration of GGR genes functiondirectly and/or indirectly attributable to the expression of GGR genes.It is also meant to indicate a loss, interruption, alteration of theinfectious and/or inflammasome activation of the protein productsencoded by GGR genes in mycobacteria. Preferably, “at least partiallyinactivated” relates to a loss of more than 10%, preferably more than30%, more preferably more than 50%, more preferably more than 70%, mostpreferably more than 90% of the function directly and/or indirectlyattributable to the expression of a GGR gene in mycobacteria. The “atleast partially inactivation” may also include a inactivation of thefull (100%) function of a GGR gene.

Partial and/or full inactivation of genes in mycobacteria for thepurpose of the present invention can be achieved by any conventionalmethod known in the art of gene mutation, e.g., recombinant insertion,transposon insertion, replacement, deletion, frameshift, anti-senseconstruct and/or homologous recombination. Preferably, the specificmethods for partially and/or fully inactivating a GGR gene inmycobacteria is to delete more than 10%, preferably more than 30%, morepreferably more than 50%, more preferably more than 70%, most preferablymore than 90% of a GGR gene sequence.

The mechanism thereof is not vital to the invention, but typically mayinvolve reduction or loss of transcription and/or translation of theaffected gene, or only transcription/translation of a dysfunctional geneproduct. Genetically engineered mutations in a gene need not necessarilybe in an exon or open reading frame, they can be in an intron or even beupstream of the start codon, as long as it affects the levels of thefunctional gene product. This can easily be checked, e.g. by Q-PCR forgene transcription levels. Functionality of a gene product can also bechecked, as one of skill in the art would know, e.g. by providing anatural substrate to the affected enzyme. Of course, for the purpose ofthe present invention, the inactivation and the extent of inactivationcan best be determined in direct comparison to a wild-type mycobacteriumor a mycobacterium of the same strain under identical experimentalconditions but comprising a full functional and expressed GGR gene.

The term “contiguous portions of a sequence” as used herein refers to anon-interrupted sequence of nucleic acids or amino acids also occurringin the same order in the sequence referred to. Particularly envisagedare contiguous portions having a length of at least 25%, 50%, 70%, 75%,80% or 90% of the length of the reference sequence, and contiguousportions are typically at least 25 nucleic acids or at least 8 aminoacids.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Nucleic acid andprotein sequence identities can be evaluated by using any method knownin the art. For example, the identities can be evaluated by using theBasic Local Alignment Search Tool (“BLAST”). The BLAST programs identityhomologous sequences by identifying similar segments between a queryamino or nucleic acid sequence and a test sequence which is preferablyobtained from protein or nuclei acid sequence database. The BLASTprogram can be used with the default parameters or with modifiedparameters provided by the user.

The term “percentage of sequence identity” is calculated by comparingtwo optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G) or the identical amino acid residue (e.g., Ala,Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp,GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 25% sequenceidentity. Alternatively, percent identity can be any integer from 25% to100%. More preferred embodiments include at least: 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a referencesequence using the programs described herein; preferably BLAST usingstandard parameters, as described. These values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like.

The term “substantial identity” of amino acid sequences for purposes ofthis invention normally means polypeptide sequence identity of at least40%. Preferred percent identity of polypeptides can be any integer from40% to 100%. More preferred embodiments include at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%.

Mycobacterium Mutants

According to a first aspect, a mycobacterium mutant is providedcomprising at least one genetically engineered mutation in at lease onegene sequence encoding global gene regulators (GGRs), wherein the GGRgene is at least partially inactivated.

In some embodiments, the mycobacterium is selected from the groupconsisting of mycobacterium avium subspecies paratuberculosis (M. ap),mycobacterium bovis (M. bovis), mycobacterium tuberculosis (M.tuberculosis), Mycobacterium avium subsp. avium (M. avium), and mixturesthereof. Preferably, the mycobacterium is M. ap.

In preferred embodiments, the GGR gene is selected from the groupconsisting of sigH, sigL, sigE, ECF-1, and mixtures thereof.

To inactivate a GGR gene in mycobacteria, one may partially orcompletely delete the sequence of the GGR gene. For example, one mayinsert heterologous sequences into the GGR gene. One may also replace aportion of the GGR gene or allele, or a majority of the GGR gene orallele, with a heterologous sequence, or an engineered (e.g., manuallyaltered, disrupted, or changed), non-functional, copy of the wild-typesequence. Preferably, the mutation is done by deleting at least about10%, at least about 20%, at least about 30%, at least about 50%, atleast about 70% or at least 90% of the wild-type GGR gene sequence. Morepreferably, the mutation is done by deleting the full GGR sequence. Insome other embodiments, the mutation may even include a deletion of morethan 100% of the wild-type GGR gene sequence (e.g., the full wild-typegene sequence may be deleted but also include one or more heterologousand/or non-functional nucleic acid sequences attached thereto orinserted therein).

While these genes across different species of mycobacteria are expected,not all genes in each species have the identical sequences, locations,or functions. But based on sequence homology, one skilled person wouldbe able to readily identify the corresponding (or equivalent) gene inother species. In a particular embodiment, the mycobacterium is M.paratuberculosis strain which has 19 GGRs, as listed in Table 1 below.

TABLE 1 List of 19 sigma factors (global gene regulators, GGR) in M.paratuberculosis genome. Gene Start End Start End Old Revised lengthnucleotide nucleotide nucleotide nucleotide Gene locus tag* locus tag**(bp) number* number* number** number** sigA MAP2820 MAPK_0949 15303148633 3150162 1062326 1060797 sigB MAP2826 MAPK_0942 999 31532583154256 1057701 1056703 sigC MAP1814 MAPK_1954 1380 1991733 19931122219261 2217882 sigD MAP4275 MAPK_4277 597 4742530 4743126 47453394745935 sigE MAP2557c MAPK_1211 756 2874942 2875697 1335260 1336015 sigFMAP3406c MAPK_0362 789 1613298 1614107 428572 429360 sigF-like MAP1474cMAPK_2294 810 3781604 3782392 2596880 2597689 sigG MAP3621c MAPK_01471101 4018733 4019833 191134 192234 sigH MAP3324c MAPK_0444 807 36929293693735 517229 518035 sigI MAP0170 MAPK_3598 858 173736 174593 40372494036392 sigJ MAP3446c MAPK_0322 888 3825108 3825995 384969 385856 sigLMAP4201 MAPK_4203 546 4670740 4671285 4673550 4674095 sigM MAP4337MAPK_4339 588 4818485 4819072 4821294 4821881 ECF-1 MAP0946c MAPK_2822924 975766 976689 3234298 3235221 ECF-2 MAP1770c MAPK_1998 936 19327831933718 2277276 2278211 ECF-3 MAP2166 MAPK_1602 891 2397944 23988341813051 1812161 ECF-4 MAP1757c MAPK_2011 630 1919501 1920130 22908642291493 ECF-5 MAP4114c MAPK_4116 1263 4584803 4586065 4588877 4587615ECF-6 MAP4217 MAPK_4219 540 4688035 4688574 4690845 4691384 *Annotationsaccording to Li, et al., 2005 (35). **Improved annotations according tothe revised genome sequence of M. paratuberculosis (GenBank AccessionNo. AE016958) (38). Bolded genes were up-regulated during macrophagesurvival and are potential vaccine mutants.

In the present invention, one of the preferred GGR genes is sigH. Theterm “sigH” as used herein refers to a gene which encodes a RNApolymerase sigma-H factor. In some embodiments, the sigH is the sigH(SEQ ID NO: 1) of M. paratuberculosis. It has a size of 807 base pairs(bp) and is located at positions 517229 to 518035 of the M.paratuberculosis genomic sequence. In one specific embodiment, amycobacterium mutant comprises at least one mutation in M. ap sigH (SEQID NO: 1) or contiguous portions thereof, or sequences at least 90%, atleast 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 orthe contiguous portions thereof. Preferably, the mutation in sigH is atleast partial deletion of the sigH gene, as characterized as “M.paratuberculosis ΔsigH”. As a result of the deletion, the sigH gene isexpressed at levels lower than the corresponding wild-type gene, or notexpressed at all. Preferably, the expression level of sigH gene is solow as to have no effect or the expressed protein is non-functional.

Another preferred GGR gene is sigL (SEQ ID NO:2). The term “sigL” asused herein refers to a gene which encodes a Sigma-L-dependenttranscriptional regulator. In some embodiments, the sigL is the sigL(SEQ ID NO:2) of M. paratuberculosis. It has a size of 546 base pairs(bp) and is located at positions 4673550 to 4674095 of the M.paratuberculosis genomic sequence. In one specific embodiment, amycobacterium mutant comprises at least one mutation in sigL (SEQ ID NO:2) or contiguous portions thereof, or sequences at least 90%, at least95%, at least 98%, or at least 99% identical to SEQ ID NO: 2 or thecontiguous portions thereof. Preferably, the mutation in the sigL geneis at least partial deletion of the sigL gene, as characterized as “M.paratuberculosis ΔsigL”. As a result of the deletion, the sigL gene isexpressed at levels lower than the corresponding wild-type gene, or notexpressed at all. Preferably, the expression level of sigL gene is solow as to have no effect or the expressed protein is non-functional.

Another preferred GGR gene is sigE (SEQ ID NO:3). The term “sigE” asused herein refers to a gene which encodes a RNA polymerase sigma-Efactor. In some embodiments, the sigE is the sigE (SEQ ID NO:3) of M.paratuberculosis. It has a size of 756 base pairs (bp) and is located atpositions 1335260 to 1336015 of the M. paratuberculosis genomicsequence. In one specific embodiment, a mycobacterium mutant comprisesat least one mutation in sigE (SEQ ID NO: 3) or contiguous portionsthereof, or sequences at least 90%, at least 95%, at least 98%, or atleast 99% identical to SEQ ID NO: 3 or the contiguous portions thereof.Preferably, the mutation in sigE gene is at least partial deletion ofthe sigE gene, as characterized as “M. paratuberculosis ΔsigE”. As aresult of the deletion, the sigE gene is expressed at levels lower thanthe corresponding wild-type gene, or not expressed at all. Preferably,the expression level of sigE gene is so low as to have no effect or theexpressed protein is non-functional.

Another yet preferred GGR gene is ECF-1. The term “ECF-1” as used hereinrefers to a gene which encodes a RNA polymerase ECF sigma factor. Insome embodiments, the ECF-1 is the ECF-1 (SEQ ID NO:4) of M.paratuberculosis. It has a size of 924 base pairs (bp) and is located atpositions 3234298 to 3235221 of the M. paratuberculosis genomicsequence. In one specific embodiment, a mycobacterium mutant comprisesat least one mutation in ECF-1 (SEQ ID NO: 4) or contiguous portionsthereof, or sequences at least 90%, at least 95%, at least 98%, or atleast 99% identical to SEQ ID NO: 4 or the contiguous portions thereof.Preferably, the mutation in ECF-1 gene is at least partial deletion ofthe ECF-1 gene, as characterized as “M. paratuberculosis Δecf-1”. As aresult of the deletion, the ECF-1 gene is expressed at levels lower thanthe corresponding wild-type gene, or not expressed at all. Preferably,the expression level of ECF-1 gene is so low as to have no effect or theexpressed protein is non-functional.

Another preferred GGR gene is sigB. The term “sigB” as used hereinrefers to a gene which encodes a RNA polymerase sigma-B factor. In someembodiments, the sigB is the sigB (SEQ ID NO:5) of M. paratuberculosis.It has a size of 999 base pairs (bp) and is located at positions 3153258to 3154256 of the M. paratuberculosis genomic sequence. In one specificembodiment, a mycobacterium mutant comprises at least one mutation insigB (SEQ ID NO: 5) or contiguous portions thereof, or sequences atleast 90%, at least 95%, at least 98%, or at least 99% identical to SEQID NO: 5 or the contiguous portions thereof. Preferably, the mutation insigE gene is at least partial deletion of the sigB gene, ascharacterized as “M. paratuberculosis ΔsigB”. As a result of thedeletion, the sigB gene is expressed at levels lower than thecorresponding wild-type gene, or not expressed at all. Preferably, theexpression level of sigB gene is so low as to have no effect or theexpressed protein is non-functional.

Also, in some embodiments, a mycobacterium mutant comprises acombination of two or more of these genes that are mutated. Forinstance, both sigH and sigL genes are mutated, or sigH gene and one ortwo of sigL, sigE, sigB and ECF-1 genes are mutated. Additionally and/oralternatively, one or more of these genes may contain more than onemutation.

In some other embodiments, a mycobacterium mutant may have furthernon-GGR mutations. One example of these further mutations includesadditional mutations in other genes that function independent ordependent from GGRs in directly or indirectly causing mycobacteriainfections. Other non-limiting examples of the further mutation sites,as described in the United States Patent Application Publication Number2007/0134274, include gcpE, pstA, kdpC, papA2, impA, umaA1, fabG2_2,aceAB, mbtH2, lpqP, map0834c, cspB, lipN, map1634 MAP-1, MAP-2, MAP-3,MAP-4, MAP-5, MAP-6, MAP-7, MAP-8, MAP-9, MAP-10, MAP-11, MAP-12,MAP-13, MAP-14, MAP-15, MAP-16, MAP-17, or MAP-18 of M.paratuberculosis, or homologs of these genomic islands, and alsoinclude, as described in the United States Patent ApplicationPublication Number 2007/0134708, MAV-1, MAV-2, MAV-3, MAV-4, MAV-5,MAV-6, MAV-7, MAV-8, MAV-9, MAV-10, MAV-11, MAV-12, MAV-13, MAV-14,MAV-15, MAV-16, MAV-17, MAV-18, MAV-19, MAV-20, MAV-21, MAV-22, MAV-23,or MAV-24, or homologs thereof.

According to the second aspect of the present invention, a mycobacteriummutant may be defined at the protein (amino acid) level. Thus, thepresent invention provides an isolated mycobacterial protein, which isencoded by the gene sequence of a mycobacterium mutant as describedherein. The protein expressed by the mutant may have a lower activityand/or function as compared to the activity of the expressed proteinfrom the wild-type strain (e.g., M. ap). In some embodiments, theactivity and/or the function of the protein is reduced by 10%, 30%, 50%,70%, 90% or more.

Vaccine

The third aspect of the present invention provides a vaccine based onthe mycobacterium mutants described herein. Specifically, in someembodiments, the mycobacterium mutant comprises at least one mutation inat lease one gene sequence encoding global gene regulators (GGRs)described herein, wherein the GGR gene is at least partiallyinactivated. Preferably, the mycobacterium is selected from the groupconsisting of Mycobacterium avium subspecies paratuberculosis (M. ap),Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M.tuberculosis), and mixtures thereof. More preferably, the mycobacteriumis M. ap.

The GGR gene may be selected from the group consisting of a sequence ofor sequence substantially identical to sigH (SEQ ID NO:1), sigL (SEQ IDNO:2), sigE (SEQ ID NO:3), ECF-1 (SEQ ID NO:4), sigB (SEQ ID NO:5) andmixtures thereof.

The vaccine of the present invention may be a suspension of liveattenuated, or inactivated (killed) microorganisms comprising themutants described herein. The types microorganisms may vary, such asviruses, bacteria, or rickettsiae of the microorganisms, or of otherantigens such as antigenic proteins and other substances derived fromthem, administered for prevention, amelioration, or treatment ofmycobacterium infections.

In some embodiments, the vaccine is a live attenuated vaccine, whereinthe mycobacterium mutant or the organism thereof used for the inventionis alive. By “alive”, we mean the mutant or the organism thereof iscapable of propagation in a subject, in particular in a mammaliansubject. To be an effective live attenuated vaccine, the livemycobacteria must be attenuated to a degree not harmful to a subject inneed thereof. Hence, the mycobacterium mutant used for the invention ispreferably non-virulent, i.e. the genes responsible for virulence havebeen inactivated, and does not evoke or at least evokes minor diseasesymptoms of a mycobacterial infection in a subject.

In some embodiments, the vaccine is an inactivated vaccine, wherein themycobacterium mutant or the organism thereof used for the invention iskilled or inactivated. The advantage of an inactive vaccine is safety.The vaccine may be inactivated by any art-known method. For example, itmay be inactivated by using chemical methods such as beta-propiolactone,thimerosal or (another mercury donating agent), formaldehyde, etc. Thevaccine may be inactivated by applying physical methods such as heat,UV-light, micro-waves etc. The vaccine may also be inactivated by usingbiological methods such as enzyme-based methods to kill or inactivatethe bacteria and any other method as is commonly applied in the art.

In some other embodiments, the vaccine is an acellular vaccine, i.e., acell-free vaccine prepared from the purified mycobacterial components ofcell-free microorganisms, carrying less risk than whole-cellpreparations.

Vaccination may be accomplished by administering nucleic acid sequenceof the mycobacterium mutant in a form of a DNA vaccine. A “DNA vaccine”or “immunogenic” or “immunological composition” is composed of at leastone vector (e.g., plasmid) which may be expressed by the cellularmachinery of the subject to be vaccinated or inoculated and of apharmaceutically acceptable carrier, vehicle, or excipient. Thenucleotide sequence of this vector encodes one or more immunogens, suchas proteins or glycoproteins capable of inducing, in the subject to bevaccinated or inoculated, a cellular immune response (mobilization ofthe T lymphocytes) and an immune response.

The vaccine of the present invention may be administered to a subject atrisk of being infected by mycobacteria, or to a subject who has beenexposed to mycobacteria, or even to a subject known to be infected withmycobacteria. The typical subject is illustratively a living organismcapable of mounting an immune response to challenge from the vaccine.Non-limiting examples of a subject include a human, any lower primate,cow, camel, dog, cat, rabbit, rat, mouse, guinea pig, pig, hamster,horse, donkey, cattle, opossum, badger, goat, sheep, or other mammals ornon-mammals.

The vaccine of the present invention is suitable for the prophylaxisand/or treatment of a disease or medical condition affected by antigenand/or immunogen expression of the mycobacterium, more preferably theprophylaxis and/or treatment of mycobacterial infections, preferably aninfection of M. ap, M. avium, M. tuberculosis, M. bovis, or mixturesthereof. According to most specific embodiments, the vaccine is avaccine against Johne's disease caused by mycobacterial infection.

The vaccine of the present invention may be administered in anyconventional dosage form in any conventional manner. In someembodiments, routes of administration include, but are not limited to,intravenously, intramuscularly, subcutaneously, intranasally,intrasynovially, by infusion, sublingually, transdermal, orally,topically, or by inhalation. The preferred modes of administration aresubcutaneous, intravenous and intranasal.

In some embodiments, the mycobacterium mutant of the vaccine may beadministered alone or in combination with adjuvants that enhancestability and/or immunogenicity of the mycobacteria, facilitateadministration of pharmaceutical compositions containing them, provideincreased dissolution or dispersion, increase propagative activity,provide adjunct therapy, and the like, including other activeingredients.

In some embodiments, the pharmaceutical dosage forms of the vaccineaccording to the present invention may include pharmaceuticallyacceptable carriers and/or adjuvants known to those of ordinary skill inthe art. These carriers and adjuvants include, for example, ionexchangers, alumina, aluminium stearate, lecithin, serum proteins,buffer substances, water, salts, electrolytes, cellulose-basedsubstances, gelatine, water, petrolatum, animal or vegetable oil,mineral or synthetic oil, saline, dextrose or other saccharide andglycol compounds such as ethylene glycol, propylene glycol orpolyethylene glycol, antioxidants, lactate, etc. Preferred dosage formsinclude tablets, capsules, solutions, suspensions, emulsions,reconstitutable powders and transdermal patches. Methods for preparingdosage forms are well known in the art.

In other embodiments, the dosage regimen of the vaccine may also bedetermined by the skilled person using his expertise (e.g. singleadministration, repeated administration (twice or more at regular orirregular intervals), etc. This will typically also depend on thedisease to be treated and the subject receiving the treatment. In atypical animal vaccination, a dose of 10⁵-10¹⁰ cfu/animal is given viaparental or oral routes of vaccination. In one embodiment, the dose isin the range of 10⁷-10¹⁰ cfu/animal. In another embodiment, the dose isin the range of 10⁵-10⁹ cfu/animal.

Differentiate Infected from Vaccinated Animals (DIVA)

In its fourth aspect, the present invention provides a method of invitro differentiating between subjects that have been infected withmycobacterium and subjects that have not been infected withmycobacterium or have been vaccinated with a mycobacterium vaccine. Inone embodiment, the method comprises the steps of (a) identifying a genesequence that is specific to a mycobacterium mutant and is not found inthe wild-type mycobacterium strain; and (b) detecting the presence orthe quantity of the sequence in the subject. In other embodiments, themethod comprises the steps of (a) identifying a gene sequence that isspecific to a wild-type mycobacterium strain and is not found in themycobacterium mutant; and (b) detecting the presence or the quantity ofthe sequence in the subject. The mycobacterium mutant for theembodiments is any one mycobacterium mutant described herein. Thesesequences are detectable by use of a complimentary probe or primer.

The gene or the polynucleotides of the invention may contain less thanan entire microbial genome and can be single- or double-stranded nucleicacids. A polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemicallysynthesized RNA or DNA or combinations thereof. The polynucleotides canbe purified free of other components, such as proteins, lipids and otherpolynucleotides. For example, the polynucleotide can be 50%, 75%, 90%,95%, 96%, 97%, 98%, 99%, or 100% purified. The purified polynucleotidescan comprise additional heterologous nucleotides (that is, nucleotidesthat are not from mycobacterium). The purified polynucleotides of theinvention can also comprise other nucleotide sequences, such assequences coding for linkers, primer, signal sequences, TMR stoptransfer sequences, transmembrane domains, or ligands.

The gene or the polynucleotides of the invention can be isolated. Anisolated polynucleotide is a naturally occurring polynucleotide that isnot immediately contiguous with one or both of the 5′ and 3′ flankinggenomic sequences with which it is naturally associated. An isolatedpolynucleotide can be, for example, a recombinant DNA molecule of anylength, provided that the nucleic acid sequences naturally foundimmediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolatedpolynucleotides also include non-naturally occurring nucleic acidmolecules.

The gene or the polynucleotides of the invention can also comprisefragments that encode immunogenic polypeptides. Polynucleotides of theinvention can encode full-length polypeptides, polypeptide fragments,and variant or fusion polypeptides. Polynucleotides of the invention cancomprise coding sequences for naturally occurring polypeptides or canencode altered sequences that do not occur in nature. If desired,polynucleotides can be cloned into an expression vector comprisingexpression control elements, including for example, origins ofreplication, promoters, enhancers, or other regulatory elements thatdrive expression of the polynucleotides of the invention in host cells.

The gene or the polynucleotides of the invention can be detected by, forexample, a probe or primer, for example, a PCR primer, or can be thebasis for designing a complimentary probe or primer, to detect thepresence and/or quantity of mycobacterium in a subject, such as abiological sample. Probes are molecules capable of interacting with atarget nucleic acid, typically in a sequence specific manner, forexample, through hybridization. Primers are a subset of probes that cansupport an specific enzymatic manipulation and that can hybridize with atarget nucleic acid such that the enzymatic manipulation occurs. Aprimer can be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art that do not interfere withthe enzymatic manipulation. “Specific” means that a gene sequencerecognizes or matches another gene of the invention with greateraffinity than to other non-specific molecules. Preferably, “specificallybinds” or “specific to” also means a gene sequence recognizes andmatches a gene sequence comprised in a wild-type mycobacterium or amycobacterium mutant described herein, with greater affinity than toother non-specific molecules. More preferably, the probe or the primeris complimentary to a mycobacterium mutant with at least one mutation inthe gene of sigH (SEQ ID NO: 1), sigL (SEQ ID NO:2), sigE (SEQ ID NO:3),ECF-1 (SEQ ID NO:4), sigB (SEQ ID NO:5) and mixtures thereof.

The hybridization of nucleic acids is well understood in the art.Typically a primer can be made from any combination of nucleotides ornucleotide derivatives or analogs available in the art. The ability ofsuch primers to specifically hybridize to mycobacterium polynucleotidesequences will enable them to be of use in detecting the presence ofcomplementary sequences in a given subject. The primers of the inventioncan hybridize to complementary sequences in a subject such as abiological sample, including, without limitation, saliva, sputum, blood,plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, woundexudate, or tissue of the subject. Polynucleotides from the sample canbe, for example, subjected to gel electrophoresis or other sizeseparation techniques or can be immobilized without size separation.

The probes or the primers can also be labeled for the detection.Suitable labels, and methods for labeling primers are known in the art.For example, the label includes, without limitation, radioactive labels,biotin labels, fluorescent labels, chemiluminescent labels,bioluminescent labels, metal chelator labels and enzyme labels. Thepolynucleotides from the sample are contacted with the probes or primersunder hybridization conditions of suitable stringencies. Preferably, theprimer is fluorescent labeled. Also, the detection of the presence orquality of the gene sequence of interest can be accomplished by anymethod known in the art. For instance, the detection can be made by aDNA amplification reaction. In some embodiments, “amplification” of DNAdenotes the use of polymerase chain reaction (PCR) to increase theconcentration of a particular DNA sequence within a mixtures of DNAsequences.

Preferably, the amplification of DNA is done by the loop-mediatedisothermal amplification (LAMP). LAMP was developed by Notomi, et al.(72), incorporated by reference herein in its entirety. Similar to PCR,LAMP utilizes a polymerization-based reaction to amplify DNA fromexamined samples, but the enzyme for LAMP, Bst DNA polymerase largefragment, possesses a DNA strand displacement activity. This makes theDNA extension step possible without having to fully denature DNAtemplates. Moreover, the primers are designed in a way that a hairpinloop structure is formed in the first cycle of amplification, and thefollowing products are further amplified in an auto-cycling manner.Therefore, in about an hour, the repeated reactions can amplify by ˜10⁹copies of DNA molecules and can be done at a constant temperature in asingle heat block, instead of at various cycles of temperature in arelatively expensive thermal cycler.

The detection of LAMP can be made by vary technologies known in the art.Preferably, the detection of LAMP can occur without gel electrophoresisthrough the addition of fluorophore (73,74). This detection method couldreadily be used in the field to detect positive samples. As a result, acomplete diagnosis can be done and visualized in about one hour (72).Quantification and multiplexing techniques have also been developedwhich could help determine the bacterial load of a sample (73,74). Inaddition, variants of Bst polymerase and primer additions have increasedBst polymerase stability, decreased amplification time, and provided thepolymerase with the ability to reverse transcribe and amplify RNAtargets (73,74). Specifically, the increase in stability could helptransfer the diagnostic process to the field.

Accordingly, the detection of the presence of the gene or the specificbinding between the gene in mycobacterium mutant and a gene that is nota component of a subject's immune response to a particular vaccine mayindicate a natural or experimental mycobacterium infection. For example,the absence of such binding or presence may indicate the absence ofmycobacterium infection. Or, a second, separate gene, such as a mutatedmycobacterium gene that specific to a component of a subject's immuneresponse to a particular mycobacterium vaccine, may be used to detect acorresponding antibodies produced in response to vaccination. Thus, ifan antibody specific to a gene in mycobacterium vaccine is detected,then the subject has been vaccinated and/or infected. The detection ofneither genes indicates no infection and no vaccination. As such,various combinations can lead to a determination of the vaccinationand/or infection status of the subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, useful methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the invention will be apparent from the detaileddescription and from the claims.

EXAMPLES

The following examples set forth preferred markers and methods inaccordance with the invention. It is to be understood, however, thatthese examples are provided by way of illustration and nothing thereinshould be taken as a limitation upon the overall scope of the invention.

Example 1

The disclosure of Example 1 has been published in Infection andImmunity, June 2013, 81(6): 2242-2257, entitled “Key Role for theAlternative Sigma Factor, SigH, in the Intracellular Life ofMycobacterium avium subsp. paratuberculosis during Macrophage Stress,”which is incorporated herein by reference in its entirety.

Bacteria.

Escherichia coli DH5α and HB101 were used as host cells for cloningpurposes in all experiments presented here. M. avium subsp.paratuberculosis K10 and Mycobacterium smegmatis mc²155 strains weregrown in Middlebrook 7H9 broth and on Middlebrook 7H10 plates aspreviously described (1).

Construction of the ΔsigH Mutant.

A specialized transduction protocol was adopted with a few modificationsto delete the sigH/MAP3324c gene using the M. avium subsp.paratuberculosis strain (12). Briefly, two ˜900-bp PCR fragmentsflanking each end of the sigH coding region were amplified and clonedinto the pYUB854 shuttle vector. The resulting pYUB854::sigHallelic-exchange substrate (AES) was then digested with PacI and ligatedto the PacI-digested concatemers of a temperature-sensitive phasmid,phAE87. The ligation mixture was then packaged into phage particles withan in vitro lambda-packaging system (GIGAPackIII; Stratagene, La Jolla,Calif.). Mid-log-phase Escherichia coli culture was transduced with thepackaged phage particles, resulting in hygromycin-resistant colonies.From the mixture of these colonies, shuttle plasmid DNA was extractedand then electroporated into M. smegmatis competent cells. Lysate ofplaques formed at 30° C. from the transformants was collected,propagated, and titrated in M. smegmatis to produce a high-titerrecombinant phage stock. A mid-log-phase culture of M. avium subsp.paratuberculosis was transduced with the phage stock at nonpermissivetemperature (37° C.) with a multiplicity of infection of 10. Individualhygromycin-resistant colonies were picked and grown in broth mediumfollowing gDNA isolation. The genotype of sigH-deletion mutants wasconfirmed with PCR and sequence analysis as outlined before (12).

Stress Treatments of M. avium Subsp. paratuberculosis.

Wild-type and the ΔsigH mutant of M. avium subsp. paratuberculosis weregrown to late-log phase (optical density at 600 nm [OD₆₀₀]=1.0), and 200ul was spread on 7H10 agar plates (DIFCO, Sparks, Md.) supplemented with0.5% glycerol, 2 ug/ml mycobactin J, and 10% ADC (2% glucose, 5% bovineserum albumin [BSA] fraction V, and 0.85% NaCl). For disc diffusionassay (DDA), 20 ul of diamide solution (0.5 M, 1 M, or 1.5 M) and H₂O₂solution (50 mM, 100 mM, or 0.5 M) was impregnated onto each 6-mm disc(Whatman, Piscataway, N.J.), and discs were placed on each of the spreadplates. As a positive control, ethambutol discs (5 ug/disc, SENSI-DISC;BD Diagnostics) were used. Plates were incubated at 37° C. until a thickconfluent lawn developed. The sustained effect of stressors (diamide andheat shock) on the viability of the wild type and ΔsigH mutant wasmonitored by determining their CFU counts. Aliquots of M. avium subsp.paratuberculosis cultures 1, 3, and 7 days following continuous exposureto 10 mMdiamide or a 45° C. water bath were serially diluted and plated.In another experiment, M. avium subsp. paratuberculosis cultures frommidlog phase were exposed to 10 mM diamide for 3 h. The cultures werecentrifuged (3,000×g, 4° C., 10 min), and pellets were immediatelystored at −80° C. until RNA extraction.

Mouse Infections.

For the animal infections with M. avium subsp. paratuberculosis strains,female BALB/c mice (Harlan Laboratories, Indianapolis, Ind.) werepurchased at 4 weeks of age and housed in a pathogen free environmentaccording to the protocol approved by the Institutional Animal Care andUse Committee, University of Wisconsin-Madison. Two groups of mice (n=15per group) were challenged intraperitoneally with the wild-type andΔsigH mutant strains of M. avium subsp. paratuberculosis. Actualinfection inoculum sizes (˜2×10⁸ CFU per mouse) of these two strainswere similar, as determined by plate count on the day of infection.Mouse groups (n=5) were sacrificed at 3, 6, and 12 weeks postinfection(wpi), and samples from livers, intestines, and spleens were collectedfor bacterial CFU enumeration and histopathological examinations asdescribed before (17). Portions of livers, spleens, and intestines werefixed in 10% neutral buffered formalin before being sectioned andstained with hematoxylin and eosin (H&E) and Ziehl-Neelsen stain.Student's t test and Mann-Whitney test were used to statisticallyevaluate differences in CFU counts among mouse groups infected with thewildtype and ΔsigH mutant strains of M. avium subsp. paratuberculosis.

Bovine Blood Monocyte Isolation and Infection.

Blood was collected from a Johne's disease-free herd that we maintainedat the University of Wisconsin-Madison. Three cows (36-month-oldHolstein, designated animals 5695, 5970, and 6117) were bled by jugularvenipuncture using blood collection bags (TERUFLEX, Somerset, N.J.)containing citrate phosphate dextrose adenine as an anticoagulant. Bloodwas transferred to 50-ml polypropylene tubes and centrifuged at 1,400×gfor 20 min at 25° C. Buffy coat containing white blood cells wasisolated and mixed with phosphate-buffered saline (PBS) (Ca²⁺- andMg²⁺-free) to a final volume of 30 ml. The cell suspension was layeredonto 58% isotonic PERCOLL density centrifugation media (Sigma) at a 1:1ratio and centrifuged at 2,000×g for 30 min at 25° C. Peripheral bloodmononuclear cells (PBMC) were collected from the PERCOLL densitycentrifugation media—PBS interface and washed three times with PBS toremove residual PERCOLL density centrifugation media. To isolate bovinemonocyte-derived macrophages (MDMs), PBMC were resuspended in RPMI 1640(Sigma-Aldrich, St. Louis, Mo.) with 20% autologous serum andtransferred to TEFLON polytetrafluoroethene jars followed by incubationfor 4 days at 37° C. and 5% CO2. MDM cells were harvested, washed, andseeded with 2×10⁶ cells/well in 24-well plates with 5% autologous serum.Immediately before MDM cell infection, M. avium subsp. paratuberculosiscultures grown to mid-log phase (OD₆₀₀ of 0.4 to 0.6) were pelleted andresuspended in an appropriate volume of cell culture medium to achieve a50:1 multiplicity of infection (MOI). The cells were incubated at 37° C.with 5% CO2 for 3 h, and, subsequently, the monolayers were washed twotimes with warm PBS to remove extracellular bacteria, and RPMI 1640medium containing 5% autologous serum was added. The plates wereincubated at 37° C. for up to 8 days, and the culture medium wasreplaced with fresh medium at 4 days after infection. In another set ofexperiments, MDM cells were pretreated overnight (18 h) with 0.01 ug/mlrecombinant bovine IFN-γ (Kingfisher Biotech, St. Paul, Minn.) beforeinfection with M. avium subsp. paratuberculosis strains. Thisconcentration of IFN-γ was adequate to activate bovine monocytes (23).Bovine MDM cells were lysed at 1, 4, and 8 days postinfection for CFUplating with serial dilutions. Student's t test was used for statisticalanalysis, where P values of 0.05 were considered to be significant toevaluate differences in CFU counts.

J744A.1 Cell Culture Infection.

The mouse macrophage cells (J774A.1) were maintained in RPMI 1640(Sigma-Aldrich, St. Louis, Mo.) supplemented with 5 to 10%heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) in 75-cm²filter-cap tissue culture flasks (Techno Plastic Products, Trasadingen,Switzerland) in a water-jacketed incubator (Thermo Scientific, Waltham,Mass.) at 37° C. with 5% CO₂. When confluent, cells were detached with acell scraper and resuspended, and 10% of the cell suspension wasreplenished with fresh culture medium every 3 to 4 days.

Macrophages were seeded at 1.5×10⁷ cells per 15-mm cell culture dish(Techno Plastic Products) in 15 ml of culture medium as described above,30 to 36 h prior to infection, and were incubated at 37° C. with 5% CO₂.At least 5 dishes were seeded for each time point. For IFN-γ activationexperiments, old culture medium was discarded and 15 ml of fresh mediumwith 100 U/ml recombinant murine IFN-γ (Pepro Tech, Rocky Hill, N.J.)was added to each dish, 16 to 20 h prior to infection. An approximate109 CFU bacterial suspension from mid-log phase was mixed with 12 ml ofcell culture medium (RPMI 1640-10% FBS, mycobactin J-free) and added toeach decanted dish (MOI=50). The cells were incubated at 37° C. with 5%CO2 for 2 or 24 h before intracellular bacteria isolation. For the24-h-time-point experiments, extracellular bacteria were washed away at2 h postinfection with 15 ml of warm PBS at least five times or until novisible bacterial particles were observed under an inverted microscopeat ×400 magnification. The washed cells were replenished with 15 ml offresh cell culture medium and incubated until 24 h postinfection. Eachcondition was replicated at least twice until the quality of extractedRNA passed the criteria described below.

Immunofluorescent Staining for LAMP-1 Expression.

Culture cells grown on a circular coverslip were fixed in 2.5%paraformaldehyde for 10 min and permeated with cold methanol-acetone(1:1) at −20° C. for 5 min. A few drops of TB AuramineM(BD Diagnostics,Franklin Lakes, N.J.) were added and incubated at room temperature for10 min to stain mycobacteria. The coverslip was washed with 95% EtOHthree times and rinsed with PBS containing 0.2% saponin and 2% goatserum. Rat monoclonal antibody 1D4B against mouse LAMP-1 purchased fromthe Developmental Studies Hybridoma Bank at the University of Iowa wasdiluted to 20 ug/ml in PBS-saponin-goat serum and incubated with thefixed cells at room temperature for 1 h. The cells were washed withPBS-saponin-goat serum three times, each for 10 min. Goat antibodyconjugated with ALEXA FLUOR 633 dye against rat IgG (Invitrogen,Carlsbad, Calif.) was diluted to 10 ug/ml in PBS-saponin-goat serum andincubated with the cells for 1 h in the dark at room temperature. Thecells were then washed in the same way as described in the last step.Finally, the coverslip was mounted on a microscope slide in VECTASHIELDmounting medium (Vector Laboratories, Burlingame, Calif.) and observedwith a Nikon C1 confocal microscope system.

Phagosome pH Measurements.

Phagosome pH measurement was slightly modified from previous studies(26, 27) based on ratiometric measurements. J774A.1 cells were seeded at2×10⁵ cells per well on a 24-well cell culture plate (Techno PlasticProducts) in 0.5 ml of culture medium with or without 100 U/ml murineIFN-γ. A poly-L-lysine (Electron Microscopy Sciences, Hatfield,Pa.)-coated 12-mm circular coverslip was placed in each well beforeseeding. After overnight incubation, culture medium was replaced with0.3 ml of prewarmed fresh medium with 5 uM LYSOSENSOR Yellow/BlueDND-160 probe (Invitrogen), and the cells were incubated for 5 min at37° C. To generate an in situ pH gradient standard curve, each coverslipwas then incubated with morpholineethanesulfonic acid (MES) buffer (25mM MES, 5 mM NaCl, 115 mM KCl, and 1.2 mM MgSO4) of known pH (from 3.5to 7.0 at a 0.5 interval), in the presence of 10 uM nigericin and 10 uMmonensin, for 2 min. The coverslip was immediately mounted on a glassslide and observed under an Olympus BX51 microscope with a reflectedfluorescence system. Sixteen-bit grayscale images of two separatechannels (excitation of 365/10 nm, emission of 460/50 nm, dichroic of400 nm; excitation of 365/10 nm, emission of 540/20 nm, dichroic of 400nm; Chroma, Bellows Falls, Vt.) from each field were taken.

The processing time from sample mounting to image acquisition wascontrolled so it took no longer than 10 min for each coverslip. Imageprocessing was done with ImageJ 1.44 k (28). For each pH standard, atleast 20 individual regions of interest (ROIs) were randomly chosen, andmean intensities of each ROI from both channels were recorded. Ratios ofintensities of green (540 nm) to blue (460 nm) from the same pH standardwere then averaged, excluding values of = or >2 standard deviations (SD)from the mean. A standard curve of ratios was plotted against pH byapplying a Boltzmann equation, y=A2+(A1−A2)/{1+exp[(x−x0)/dx]}, where A1and A2 represent the limits of the fluorescent ratio at infinitely lowand high pHs, respectively, x0 is the pH midpoint at (A1+A2)/2, x is theobserved pH, and dx is the slope of the curve. When needed, cells wereinfected with M. avium subsp. paratuberculosis as described above,except the bacteria were prestained with 5 uM VYBRANT dye DiDcell-labeling solutions (Invitrogen) for 10 min (29). Intracellularbacteria could be observed with a third filter set (excitation of 535/50nm, emission of 675/20 nm, dichroic of 565 nm). ROIs were chosen wherethe bacteria were colocalized with LYSOSENSOR-stained phagosomes. Theaverage 540/460 ratio of ROIs was plugged into the equation to calculatephagosome pH.

Intracellular Bacterial Isolation and RNA Extraction.

Intracellular bacteria were isolated by a protocol described before (30)with modifications. At 2 or 24 h post infection, infected cells werewashed with 15 ml ice-cold PBS at least five times or until no visiblebacterial particles were observed under an inverted microscope. Thewashed cells on each dish were then lysed with 10 ml cell lysis buffer(4 M guanidine thiocyanate, 0.5% sodium N-lauryl sarcosine, 25 mM sodiumcitrate, 0.5% TWEEN polusorbate 80, and 0.1 M β-mercaptoethanol) andcollected with a rubber cell scraper. To reduce viscosity and helpdissolve cell debris, cell lysates from all dishes were pooled andpassed through a 23-gauge needle five times. The lysate was then splitinto four 14-ml polypropylene centrifuge tubes (FALCON 352059; BDBiosciences, San Jose, Calif.) and centrifuged at 3,200×g and 4° C. for25 min. Each pellet was washed in 1 ml of TRIZOL regent (Invitrogen)twice and subjected to RNA extraction. Total RNA was extracted by aprotocol described before (12, 31). Briefly, bacterial pellets wereresuspended in 2 ml of TRIZOL reagent and split into two 2-ml screw-captubes, each with 3.0 g of 0.1-mm zirconia/silica beads (BioSpectProducts, Bartlesville, Okla.) and disrupted in a Mini-BeadBeater-8(BioSpect Products) at top speed for three pulses of 60 s with 30-sintervals on ice. Following a 5-min incubation at room temperature, thesupernatant was transferred to RNase-free tubes and centrifuged at12,000×g for 15 min. RNA was then isolated according to themanufacturer's instruction. To remove genomic DNA contamination, RNAsamples were treated with 10 U of TURBO DNase (Ambion, Austin, Tex.) at37° C. for 30 min. An IS900 241-bp PCR was performed to confirm that nogenomic DNA was detectable in the RNA samples (1). DNase treatments wererepeated if needed. Quality of the extracted RNA was examined with aNANODROP 1000 spectrophotometer (Thermo Scientific). The ratios ofA₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ must be higher than 1.8 and 1.5, respectively,before proceeding to cDNA synthesis for transcriptome studies.

Transcriptome Studies.

The NimbleGen (NimbleGen System Inc., Madison, Wis.) M. avium subsp.paratuberculosis microarray was designed from the 4,350 open readingframe sequences in the genome of M. avium subsp. paratuberculosis (32).The whole genome was represented three times on each chip. In addition,each gene was represented by 20 probes of 60-mer oligonucleotides. As aresult, each gene was represented by a total of 60 probes.Double-stranded cDNA synthesis from isolated RNA samples, microarrayhybridization, and data analysis were performed as previously described(12, 33). Significantly expressed genes were selected by ±2-fold ofchange and P values of <0.05 by Student's t test. The intensities werealso exported to Gene Spring GX (Agilent Technologies, Santa ClaraCalif.) for principal component analysis (PCA) on treatment conditions,which is a method to reduce dimensionality in multicondition microarrayexperiments and to find relevant patterns across conditions (34). Twobiological replicates were included for each condition.

For RNA-seq studies, purified RNA samples were used for depletion ofrRNA sequences to enrich mRNA using the MICROBEXPRESS bacterial mRNAenrichment kit (Ambion, Austin, Tex.). Approximately, 10 ug of total RNAfor each sample was processed according to the manufacturer'sinstructions. For cDNA library preparation and sequencing, samplescontaining at least 1 ug of enriched mRNA were sent to the DNASequencing Facility at the University of Wisconsin-Madison BiotechnologyCenter. An Illumina HiSeq 2000 platform using one flow cell lane with100-cycle paired-end chemistry (Illumina, San Diego, Calif.) was used tosequence the cDNA library clusters. Raw RNA-seq data files in FASTQformat were assembled against the M. avium subsp. paratuberculosis (35)using the CLC Genomics Workbench 4.8 (CLC bio, Aarhus, Denmark). Geneexpression for each of the different sample conditions was calculatedusing “reads per kilobase million” (RPKM) expression values (36). Thefollowing formula was used to determine the RPKM values: RPKM=number ofreads/(kilobase length of gene×millions of mapped reads).

This RPKM metric enables comparisons between data sets with a varyingnumber of total reads. All reads mapping to annotated noncoding RNA(ncRNA) were removed from the data sets before determining RPKM values.Data sets were quantile normalized, and Kal's Z-test (37) was used forthe comparative gene expression analysis. Genes were consideredsignificantly, differentially expressed if they showed a ±2.5-foldchange with FDR P values of <0.05.

Quantitative Real-Time PCR.

Quantitative real-time PCR (qRT-PCR) was previously described (12, 38)for confirmation of transcript levels. A SYBR green-based reagent withROX (Bio-Rad, Hercules, Calif.) was used with 50 ng of double-strandedcDNA in each reaction. Double-stranded cDNA synthesis is described inthe microarray sample preparation session. No gDNA was detected from theRNA samples for cDNA synthesis. qRT-PCRs were performed with a 7300real-time PCR system (Applied Biosystems, Foster City, Calif.). Thethreshold cycle (CT) of each gene was normalized to the CT of the 16SrRNA gene from the same cDNA sample. The expression fold changes werecalculated by comparing the normalized CT of treated samples to thecontrol sample as detailed before (39, 40).

Microarray Data Accession Number.

Data sets discussed in this report were deposited in NCBI's GeneExpression Omnibus (41) and are accessible through GEO Series accessionnumber GSE43645.

Results

Characterization of M. avium subsp. paratuberculosis-ContainingPhagosomes.

In our previous study, we defined the stressome of M. avium subsp.paratuberculosis under various in vitro conditions that mimicked thehostile host microenvironments, including low pH and oxidative stress(12). In the present study, we further examined the bacterial responsesin the early stage of cell infection using a murine macrophage infectionmodel. Both naive and IFN-γ-activated cells were used in our study. Wemonitored the expression of inducible nitric oxide synthase (iNOS), amarker for macrophage activation, with quantitative real-time PCRfollowing IFN-γ treatment of J774A.1 cells. The transcription activityof iNOS in IFN-γ-treated cells was over 1,000 times higher than naivecells (data not shown). The temporal profile of iNOS expressionindicated that naive macrophages were activated by 2 h postinfection andthroughout the course of infection, with comparable mRNA levelsregardless of the viability of M. avium subsp. paratuberculosis bacilli.Activated macrophages had a similar profile, but the iNOS expressionlevels were between 1.6 to 2.6 times greater than those of infectednaive macrophages at each time point.

Additionally, we measured the phagosomal pH in both naive and activatedmacrophages using a dual-emission dye LYSOSENSOR DN-160 that emitsfluorescent signals in a pH-dependent manner. Before infection, naiveand activated macrophages had similar lysosomal pH levels ranging from5.1 to 5.3. At 2 h postinfection, the pH in phagosomes containingheat-killed M. avium subsp. paratuberculosis decreased below 4.0regardless of cell activation status. However, the pH in phagosomescontaining live M. avium subsp. paratuberculosis bacilli decreased justbelow the preinfection level (i.e., 4.8 to 5.0), suggesting the abilityof live bacteria to prevent phagosome acidification. Activatedmacrophages, but not naive ones, were able to continuously decrease thepH of phagosomes containing live bacilli up to 4 h of postinfection. Asthe infection progressed (24 h), activated macrophages exhibited abetter ability to maintain a lower pH level than naive macrophages.

To examine the role of M. avium subsp. paratuberculosis on phagosomematuration, we examined the percentage of colocalization betweenintracellular M. avium subsp. Paratuberculosis and the lysosome markerLAMP-1. While heat-killed bacteria showed over 85% colocalization withLAMP-1, live M. avium subsp. paratuberculosis significantly reduced thepercentage of colocalization with LAMP-1 at 2 h postinfection(67.6%+5.5) in naive macrophages, suggesting live M. avium subsp.paratuberculosis is able to rapidly circumvent the hostile environmentand to delay phagosome maturation. The percentage of colocalization didnot significantly change in activated macrophages over the course of theexperiment (87.92%±5.32 and 83.7%±9.5 at 2 h and 24 h, respectively),suggesting that preactivated host cells have a better ability to controlinvading intracellular pathogens by means of phagosome maturation. Thereduced phagolysosome fusion of naive macrophages was restored to alevel (78.4%±6.8) similar to that of the preactivated phagosome(81.6%±8.8) at 24 h postinfection, as also evidenced by the increasediNOS expression level of the naive macrophage infection compared touninfected cells (data not shown). In general, both phagosomal pH andLAMP-1 colocalization indicated the ability of live, virulent M. aviumsubsp. paratuberculosis to prevent phagosome acidification and to delaylysosomal fusion by 2 h postinfection.

Transcriptional Profiling of M. avium Subsp. Paratuberculosis Isolatedfrom Infected Macrophages.

To profile changes in the levels of M. avium subsp. paratuberculosistranscripts within macrophages, we isolated intracellular bacilli at 2and 24 h postinfection, with or without IFN-γ activation. Because thebacteria must stay in the cell culture medium (RPMI 1640-10% FBS,mycobactin J-free) before they can infect host macrophages, we comparedthe transcriptomes of intracellular bacteria to those incubated in vitroin cell culture medium for 2 h. Under all conditions tested, thecorrelation coefficients (r) between biological replicates rangedbetween 0.92 and 0.99. To examine the statistical distance between eachbiological replicate and among treatments, a three-dimensional principalcomponent analysis plot was generated, indicating high correlationsbetween biological replicates. Cluster analysis identified groups ofgenes active only during macrophage infection. Compared to theRPMI-incubated control sample, expression levels of 136 and 333 M. aviumsubsp. paratuberculosis genes were significantly changed in naivemacrophages at 2 and 24 h postinfection, respectively. On the otherhand, in IFN-γ-activated macrophages, the numbers of genes withsignificantly changed expression levels were 284 and 328, respectively.Among those genes, 47 were common in all of the 4 examined macrophageconditions, representing a core set of genes responsible for interactingwith the macrophage microenvironment.

In general, M. avium subsp. paratuberculosis transcriptomes in infectedmacrophages preactivated with IFN-γ were more similar to those observedunder in vitro stressors reported earlier (12). Also, IFN-γ activationof macrophages resulted in significant induction of a group of genes(n=48), mostly those involved in energy production and conversion (e.g.,icl, fdxA, sdhABCD, and ndh) or nutrient transport and metabolism (e.g.,fad genes, dapA_1, and cysH_2), at 2 h postinfection in IFN-γ-activatedmacrophages compared to naive macrophages.

At 24 h postinfection, we started to see a significant change in M.avium subsp. paratuberculosis transcriptomes indicative of change oftheir microenvironment, especially in activated macrophages. Forexample, the mbt operon (mbtA to mbtE and MAP2172c) involved in ironmetabolism was significantly upregulated at 24 h postinfection inIFN-γ-treated macrophages compared to the RPMI-incubated controlsamples. A similar observation was reported for 120 h postinfection ofbovine monocyte-derived macrophages (MDMs) (21). MAP2172c was shown tobe repressed in M. avium subsp. paratuberculosis cultures grown inmycobactin J-depleted medium over time, where expression levels of othermbt genes remained constant (42). This paradigm could suggest that anintracellular environment is not iron exochelin-free, or there are otherintracellular factors that stimulate alternative iron metabolicpathways, at least in the examined times. On the other hand, a geneinvolved in iron storage, bfrA (43), was significantly downregulated atthe same time point, while the mbt operon was activated, suggesting thelack of access to iron inside the mycobacterial phagosome.Interestingly, the ex-pression of the iron-dependent regulator ideR (43)remained unchanged during the examined time course regardless ofmacrophage activation status, suggesting a lesser role of ideR in earlystages of macrophage infection. Overall, a significant time-dependentshift in M. avium subsp. paratuberculosis transcriptomes was evidentfrom examining M. avium subsp. paratuberculosis collected at 2 and 24 hafter macrophage infection.

Microenvironment of M. avium Subsp. Paratuberculosis.

One of our goals is to gain more insights into the intracellularenvironment of M. avium subsp. paratuberculosis using transcriptomeanalysis. Schnappinger et al. reported that M. tuberculosis upregulatesβ-oxidation genes by 4 h postinfection in murine bone marrowmacrophages, suggesting a transition of carbon source from carbohydratesto fatty acids (44). Similarly, our study indicated activation of M.avium subsp. paratuberculosis orthologues (fadA6_3, fadB_1, fadD9,fadE17, fadE21, fadE3_1, and fadE5) in the β-oxidation pathway startingfrom as early as 2 h postinfection, suggesting the transition of carbonsource utilization is a common bacterial strategy between M.tuberculosis and M. avium subsp. paratuberculosis. The icl gene,previously known as aceA, was also among the highly upregulated genesinvolved in carbon metabolism. The gene product, an isocitrate lyase,bridges the β-oxidation pathway to glyoxylate cycle, an anabolic pathwaywith a net product of glucose. The contribution of icl to M. aviumsubsp. paratuberculosis survival in macrophages remains to be analyzed.

Once entering host cell compartments, intracellular bacteria encounterhost defense mechanisms such as reactive oxygen intermediates (ROIs),reactive nitrogen intermediates (RNIs), digestive enzymes, and, mostimportantly, lower pH. In addition, the phagosome is also anutrient-depleted environment. Accordingly, we examined genes that areassociated with stress response and intracellular bacterial survival.Several known oxidative stress-induced genes, such as oxyR, trxB, trxC,tpx, ahpC, and ahpD, were significantly upregulated in all intracellularconditions. OxyR is a redox sensor protein that, when oxidized,positively regulates a group of genes, including ahpC, katG, gorA, andfurA (45). Among those genes, the ahpCD operon was highly upregulated(6.2- to 11.0-fold) in samples taken from naive or activatedmacrophages. In addition, TrxB, TrxC, and Tpx, proteins involved inreduction of thio-disulfide and resistance of hydroperoxide processes(46), were upregulated, suggesting active machinery for counteractingoxidative stress within host cells. However, other known oxidativeresponsive genes, such as katG, gorA, furA, sodA, and sodC, were notactivated in these samples, possibly because those genes are indirectlyregulated by oxyR or also under the control of other stress-responseregulators. Overall, M. avium subsp. paratuberculosis deployed specificgene products to defend against the hostile microenvironment during thisearly stage of infection.

Changes in Global Gene Regulators.

Sigma factors play a central role in bacterial gene regulation (47) andpathogenesis as reviewed in M. tuberculosis (48) and other pathogens(49). Nineteen sigma factors were identified in M. avium subsp.paratuberculosis by sequence analysis (24), 13 of which are homologousto M. tuberculosis sigma factors. The sigA gene, though considered aconstitutively expressed sigma factor gene in M. tuberculosis (50), wasfound downregulated to nearly 2-fold at 24 h in activated macrophages.The sigB gene, which is a dispensable sigma factor and partiallyresponsive to some oxidative and heat shock stresses in M. tuberculosis(51), showed a slight increase (˜1.6-fold) at 24 h postinfection underboth activated and naive states of macrophages. Genes of sigC, sigG,sigJ, sigM, and other extracytoplasmic sigma factors (ECF-2 throughECF-6) remained constantly expressed in all examined conditions.However, as shown in FIG. 1, sigH was the most induced among other sigmafactors of M. avium subsp. paratuberculosis, and the activation seems tobe augmented by macrophage activation over time. sigH transcripts wereupregulated under in vitro heat shock and oxidative stress treatments inM. avium subsp. paratuberculosis (12). Also, sigL was upregulated at the2-h time point but downregulated by 24 h postinfection, suggesting apotential role for sigL in the very early stage of infection. On thecontrary, sigE expression was significantly upregulated after 2 hpostinfection and remained high at 24 h postinfection, suggesting aprolonged role during M. avium subsp. paratuberculosis persistence.Overall, a few sigma factors showed a dynamic and active gene regulationtransition during the first 24 h postinfection within macrophages. It ispossible that other regulators could play a similar role during latertimes of infection or in different host cells.

Role of sigH in M. avium Subsp. Paratuberculosis to Variable StressConditions.

The sigH gene in M. tuberculosis has been shown to be upregulated uponheat shock, upon diamide treatment (25), and during survival in humanmacrophages (52), suggesting its importance in responding toextracellular stimuli and intracellular survival. To test the hypothesisthat sigH could play an important role in M. avium subsp.paratuberculosis stress responses, we employed a specializedtransduction protocol (53) to generate a sigH isogenic knockout mutantin the M. avium subsp. paratuberculosis K10 genetic background. BecausesigH and its anti-sigma factor (MAP3323c) are likely encoded in anoperon (54), the ΔsigH mutant was examined for possible polar effects onthe downstream gene, MAP3323c. Using reverse transcriptase PCR, thepresence of the MAP3323c transcript was confirmed in the ΔsigH mutantstrain.

After construction, the resistance of the ΔsigH mutant was evaluatedagainst various stressors. Analysis of the disc diffusion assay revealedthat the ΔsigH mutant does not tolerate thiol-specific oxidationcompared to the wild-type strain, as evidenced by the observed halozones on plates. However, no other differential resistance was observedwhen a cell wall stressor (sodium dodecyl sulfate) or ethambutol discswere used (data not shown). To measure viability of the ΔsigH mutantafter sustained exposure to diamide or heat stress, we cultured bothwild-type and the mutant strains for an extended time period in thepresence of diamide or a 45° C. water bath. In both stress conditions,there was significant reduction at each time point in the viability ofthe ΔsigH mutant compared to that of the wild-type strain. At day 7, theviability of the ΔsigH mutant was reduced by almost 2-log orders andmore than 1-log order in CFU counts relative to the wild-type strain fordiamide and heat stress, respectively. Unfortunately, replicativeplasmid complementation of the ΔsigH mutant with a wild-type sigH underthe control of the hsp65 promoter did not restore the diamide resistancephenotype (data not shown), most likely due to inefficientcomplementation in mycobacterial strains (25, 55, 56).

Intracellular Survival of the ΔsigH Mutant in Bovine MDM Cells.

Intracellular growth kinetics of M. avium subsp. paratuberculosisstrains were analyzed using bovine MDM cells. MDM cells were infectedwith the ΔsigH mutant and its parental strain for a prolonged time up to8 days after infection. The MDM monolayer in the culture wells waschecked at a regular interval for cell confluence under an invertedlight microscope. We first determined intracellular viability of boththe ΔsigH mutant and wild-type strains within the naive MDM cells. Thenumbers of wild-type strain of M. avium subsp. paratuberculosis bacilliincreased 2-fold, whereas the growth of the ΔsigH mutant was notsupported within naive MDM cells as determined by CFU plating at 8 daysafter infection (FIG. 2A). Next, we examined whether the ΔsigH mutantwould be able to survive inside activated MDM cells pretreated withrecombinant IFN-γ. At 8 days post infection, there was more than a2-fold increase in the number of wild-type M. avium subsp.paratuberculosis bacilli, whereas in the IFN-γ pretreated MDM cells,viability of the ΔsigH mutant was significantly reduced almost by 50%(FIG. 2B). These observations suggested an important role for sigH indefending M. avium subsp. paratuberculosis against IFN-γ activation.

Virulence Analysis of the M. avium Subsp. Paratuberculosis ΔsigH Mutant.

To assess the role of SigH in M. avium subsp. paratuberculosisvirulence, we investigated the persistence of the M. avium subsp.paratuberculosis ΔsigH mutant using the mouse model of paratuberculosis.The initial growth kinetics of the wildtype and ΔsigH mutant strainswere similar, with an equal burden of bacteria in both intestine andspleen up to 6 wpi (FIG. 3). However, the colonization levels of theΔsigH mutant compared to its parental strain were significantly reducedin spleen and intestine at 12 wpi, suggesting a role for sigH in thelong-term survival of M. avium subsp. paratuberculosis in mice.Interestingly, when M. tuberculosis ΔsigH was used to challenge mice, nodifferences in bacterial load were observed in mouse organs compared tothat of the wild-type strain (57).

Evaluation of the hematoxylin and eosin-stained spleen, liver, andintestine organs at 3, 6, and 12 wpi showed moderately similar tissuepathology when infected with the ΔsigH mutant or wildtype M. aviumsubsp. paratuberculosis. Granulomatous inflammation was evident in theliver tissues by 12 wpi, with no visual differences in mycobacterialcolonization among the mouse groups infected with wild-type or mutantstrains. However, mouse spleen tissues infected with the wild-typestrain displayed higher follicular atrophy than the spleen tissuesinfected with the ΔsigH mutant. Consistent with the bacterialcolonization data, Ziehl-Neelsen staining showed higher numbers ofacid-fast bacilli in the mouse spleen infected with the wild-type strainthan the ΔsigH mutant at 12 wpi (FIG. 3C). Taken together, our dataindicated that the ΔsigH mutant was attenuated in the murine model ofparatuberculosis compared to the wild-type M. avium subsp.paratuberculosis strain.

Transcriptional Regulation of sigH in M. avium Subsp. Paratuberculosis.

Our stress experiments showed that the ΔsigH mutant was hypersensitiveto elevated temperature and diamide exposure, each resulting in impairedgrowth. On the basis of these findings, we hypothesized that sigH mayplay an important role in directing transcriptional control underunfavorable environmental conditions. To identify gene regulatorynetworks under the control of sigH, both wild-type M. avium subsp.paratuberculosis and ΔsigH mutant transcriptomes were profiled beforeand after diamide exposure using the next-generation RNA sequencing(RNA-seq). When the wild-type strain was compared to the ΔsigH mutant,approximately 15% of the M. avium subsp. paratuberculosis genes (˜307induced and ˜344 repressed) were found to be differentially regulated at3 h postexposure to diamide stress. This large number of geneperturbation was likely orchestrated by additional sigma factors (e.g.,sigB, sigD, sigE) along with various transcriptional regulators thatwere differentially expressed in examined samples. Genes were groupedinto different functional categories, and a large number of genes (e.g.,hsp, clpB) belonging to the chaperonin functional category weresignificantly upregulated. Many induced genes were involved indetoxification and maintaining cellular redox homeostasis (e.g., trxB2,adhE) during oxidative stress as detailed before (46, 58). Manytranscriptional factors and two-component systems were found to beupregulated (e.g., sigB, sigE, whiB4, MAPK_0206, mtrA) under the controlof SigH. The expression of mycobacterial sigB and sigE was known to belinked with the presence of the sigH gene (59). WhiB-like proteins areredox-responsive DNA binding factors and could play a protective roleagainst oxidative stress (60). In mycobacteria, the role of theMtrB-MtrA two-component system is not entirely understood, but it wasfound to be essential for bacterial viability, particularly involved inthe regulation of cell wall permeability (61, 62).

A number of induced genes in the SigH regulon were related to virulence,and many of them were included in the mce gene family (e.g., mceAl,mceC, mceD). Several mce genes were shown to be upregulated duringphagocytosis and oxidative stress exposure (52, 63), indicating thatthey are active during infection. Other key functional gene categorieswere associated with central intermediary/sulfate metabolism (e.g.,rmlB, rmlC, cysQ_2, cysD), energy metabolism (e.g., rpi, tpi, nuoA), andcell processes/transport (e.g., fdxC_2, MAPK_4062) or were cell enveloperelated (e.g., mmpL4_1, mmpS3). Our results indicate that many of thesegenes were induced under intraphagosomal stresses inside macrophages.Genes belonging to functional categories, including lipid metabolism(e.g., fadE14, fadD33_2, MAPK_2213), polyketides (e.g., pks2, papA3_2),and biosynthesis of amino acids (e.g., leuC, metA, trpE2), were amongthe down regulated genes in M. avium subsp. paratuberculosis relative tothe ΔsigH mutant following diamide stress. We also examined thedifferential expression profile of M. avium subsp. paratuberculosis inthe absence of sigH during standard physiological growth conditions(mid-log phase). In this case, gene categories belong to lipidmetabolism (e.g., fadD29), cell processes (e.g., kdpA, pstS),transcriptional regulation (e.g., MAPK_0788), and electron transport(e.g., fdxC_2). Additionally, we found that a large number of genesbelong to the hypothetical functional category (˜30%). To verify thetranscriptome results, a few upregulated genes were randomly selected,and qRTPCR was employed using the SYBR green method. The transcriptlevels of these genes analyzed by RNA-seq and qRT-PCR were in goodagreement and corroborated with the transcriptome data.

Identification of sigH-Regulated Promoters in M. avium Subsp.Paratuberculosis.

For identification of promoters that were likely to be directlycontrolled by sigH, we analyzed a list of candidate transcripts withhigher expression ratios (wild type/ΔsigH mutant). Since SigH of M.avium subsp. paratuberculosis is a very close homologue of M.tuberculosis SigH (59), we searched for the presence of the consensussequence of M. tuberculosis SigH-dependent nondegenerate promoter motifsGGAA-N18-20-GTT in the 250-bp region upstream of start codons of M.avium subsp. paratuberculosis genes using the Genolist webserver. Atotal of 30 genes were found to be directly upregulated by SigH withGGAA and GTT core motifs at the −35 and −10 regions, respectively, intheir promoter regions. Many of these targets of SigH in M. avium subsp.paratuberculosis were also found to be controlled in Corynebacteriumglutamicum (64), Streptomyces coelicolor (65), and M. tuberculosis (59),suggesting a conserved regulon directly controlled by SigH across thehigh-percentage GC Gram-positive actinobacteria.

Discussion

The intracellular pathogen M. avium subsp. paratuberculosis is known toinfect and persist within host macrophages with unclear mechanisms. Toexamine how M. avium subsp. paratuberculosis responds to intracellularenvironments, especially during the early stages of infection, we used amacrophage cell line coupled with DNA microarrays to profilemacrophage-induced changes in M. avium subsp. paratuberculosistranscriptome. A clear advantage of this infection model is theflexibility to control the activation status of the host cells inaddition to the availability of reagents and protocols for manipulation.By comparing the results of phagosome pH and phagosome colocalizationmarkers, we found significant differences in intracellular environmentsof naive versus active macrophages consistent with earlier studies (44,66). Activated macrophages, at the time of infection, showed much higheriNOS gene expression than naive macrophages. At 2 or 24 h postinfection,they showed higher phagosome colocalization with ingested M. aviumsubsp. paratuberculosis particles, which clearly exhibited a better celldefense mechanism than naive macrophages. However, during the course ofinfection up to 24 h, overall survival of intracellular M. avium subsp.paratuberculosis did not differ in either naive or activatedmacrophages. This phenotype could change later during persistentinfection which we did not address in this study. Most of thedifferentially expressed genes between these states are core stressresponsive genes involved in energy production, indicating M. aviumsubsp. paratuberculosis initiates stress responses to a higher levelmore rapidly in activated intracellular environments. On the host side,once activated, the host cells maintained their activation statusthroughout the course of infection. This suggests that virulent M. aviumsubsp. paratuberculosis has the ability to prevent phagosome maturationand subsequently circumvent detrimental low pH and oxidative stressesduring the very early stages of infection, possibly without interferingwith the host early signal transduction pathways responsible formacrophage activation.

When M. avium subsp. paratuberculosis transcriptomes in macrophages werecompared to our previous study of the in vitro stressome (12), therewere more common genes with the 24-h than 2-h-postinfection samples,indicating that during the early stage of infection, M. avium subsp.paratuberculosis is adjusting to more acidic and oxidizing environments.We also observed the metabolic shift of M. avium subsp. paratuberculosisto utilize fatty acids as the major carbon source, which has alreadybeen observed in M. tuberculosis (44) and M. avium subsp.paratuberculosis (21). The shift of metabolic activity at earlyinfection may be a common theme employed by mycobacterial pathogensunder nutrient-depleted conditions. By 24 h postinfection, securing ironfor M. avium subsp. paratuberculosis became a significant quest,especially in activated macrophages, as suggested by the activation ofthe mbt operon. It is well established that the phagosome is aniron-depleted compartment (44) and intracellular pathogens have evolvedways to scavenge iron within mammalian cells. However, iron acquisitionmechanisms of M. avium subsp. paratuberculosis remain unknown given thatM. avium subsp. paratuberculosis possesses a truncated mtbA gene andthus is unable to produce mycobactin (32).

Because of the important role played by global gene regulators inbacterial pathogenesis, we focused our analysis on the expressionprofile of the 19 sigma factors encoded in the M. avium subsp.paratuberculosis genome (32). Accordingly, in the experiments reportedhere, we were able to capture active gene regulation of a set of sigmafactors (e.g., sigH, ECF-1) during early macrophage infection with M.avium subsp. paratuberculosis. Consistent with the M. tuberculosisinfection studies, we found an immediate upregulation of sigH withinmacrophages. This trend continued through 24 h postinfection andindicated a crucial role of sigH to regulate stress-responsive genes,especially those activated during exposure to thiol oxidation, asindicated by the disc diffusion assay. Moreover, we have demonstratedthat the ΔsigH mutant is very sensitive to sustained exposure to diamideor heat stress compared to the wild-type strain. The sigE gene, anotherstress-induced sigma factor, did not show higher expression levels until24 h postinfection in either naive or activated macrophages. Thisdelayed response of sigE as well as modest induction of sigB mayindicate an indirect regulation by other immediate stress-responsivegenes and support the important role played by sigma factors inmycobacterial pathogenesis (25, 59). The other sigma factor that wasupregulated throughout the examined time course was ECF-1. This sigmafactor may also play an important role immediately upon infection, whichwas not reported before.

We have also profiled the regulatory network under the control of sigHby studying the relative abundance of gene transcripts using RNA-seq. Wefound that a large number of M. avium subsp. paratuberculosis genes weredirectly or indirectly regulated by sigH after exposure to diamidestress. In fact, analysis of the upstream sequence of the upregulatedgenes revealed a set of genes that could be directly controlled by SigH.Among them, many genes are involved in the functional category of heatshock and protein processing. Heat shock proteins (e.g., Hsp, DnaJ2,ClpB) are found widely on prokaryotic cells and act as molecularchaperones helping to configure proteins correctly upon encountering anunfavorable milieu (67, 68). Such environments, i.e., oxidative stress,could result in nonrepairable protein structures which may necessitatefull degradation by the ClpC protease (63). Oxidative stress scavengersinduced following diamide stress include TrxB2, TrxC, and AdhE (46, 58).All of these genes likely play important roles in redox homeostasisunder thiol oxidation and are found in high levels inside themacrophages (44, 63). Interestingly, the effect of diamide stress in M.tuberculosis also resulted in a transcriptional profile similar to thatof M. avium subsp. paratuberculosis (63), indicating the pivotal role ofsigH across mycobacterial species.

Consistent with the estimated large regulon of SigH, a significantdifference in survival rates between the ΔsigH and wild-type strains wasobserved inside activated bovine macrophages. Intracellular growth ofthe wild-type M. avium subsp. paratuberculosis strain was not inhibitedregardless of the activation state up to 8 days postinfection. Thisobservation was in corroboration with the earlier findings which showedthat activated bovine monocytes were inadequate to inhibit intracellulargrowth of M. avium subsp. paratuberculosis up to 9 days after infectionas determined by the CFU method (69). In contrast, viability of theΔsigH mutant was significantly impaired, indicating an importantfunction of sigH for the intracellular growth of M. avium subsp.paratuberculosis, possibly by blocking IFN-γ activity as suggestedearlier (23). In recent studies, clues have been obtained on macrophageinteraction with M. avium subsp. paratuberculosis that indicate thecapacity of this pathogen to subvert host immune responses by blockingthe ability of mononuclear phagocyte maturation (23, 70, 71). Althoughit is tempting to speculate that the ΔsigH mutant failed to interferewith macrophage maturation, especially when preactivated with IFN-γ,more experiments are needed to fully understand the mechanisms that sigHcould play during macrophage infection. The survival profile of M. aviumsubsp. paratuberculosis constructs in MDM was further supported by theinability of the ΔsigH mutant to survive in mice. Both bacteriologicaland histological analyses displayed impaired organ colonization of theΔsigH mutant with a low inflammatory response. However, we did not findany comparative differences in liver organs infected with the wild-typeand ΔsigH mutant strains. A recent study showed that the M. tuberculosisΔsigH mutant was completely attenuated in nonhuman primates (72), abetter experimental model than mice for studying human tuberculosis(57). It will be interesting and important to examine the survival ofthe M. avium subsp. paratuberculosis ΔsigH mutant in a ruminant model ofparatuberculosis (e.g., goat).

In conclusion, our analyses indicated significant changes inmycobacterial gene expression during macrophage survival, most likelyunder the control of sigH and other sigma factors. The activation statusof macrophages also directs the mycobacterial response to a specificstress-responsive profile. We demonstrated that sigH offers a massivetemporal response on the M. avium subsp. paratuberculosis transcriptometo cope with the adverse effects of oxidative stress. Our data indicatethat sigH could play a critical role during infection, and activation ofits regulon is required for replication and full virulence of M. aviumsubsp. paratuberculosis. Further interrogation of these sigma factorsand their regulatory networks should ultimately furnish a greaterunderstanding of M. avium subsp. paratuberculosis pathogenesis and helpdesign a better approach for controlling Johne's disease.

Example 2

References cited in this Example are listed in the section of Referencesas “References cited in Example 2.”

Materials and Methods

Bacterial Strains.

M. avium subsp. paratuberculosis K10 and M. smegmatis mc²155 strainswere grown in Middlebrook 7H9 broth and on Middlebrook 7H10 plates aspreviously described (2, 19). For cloning, Escherichia coli DH5α andHB101 were used as host cells. To generate an M. avium subsp.paratuberculosis mutant, a specialized transduction protocol was adoptedto delete the sigL/MAP4201 gene using the M. avium subsp.paratuberculosis K10 strain (19, 25). Primers were designed to amplify˜750 bp PCR fragments flanking each end of the sigL coding region andcloned into pYUB854 (19). The resulting vector was used to generate sigLdeleted mutant, M. avium subsp. paratuberculosis ΔsigL, according to themethods described elsewhere (26). Genotype of the M. avium subsp.paratuberculosis ΔsigL was confirmed by PCR and sequence analysis (19).To complement the M. avium subsp. paratuberculosis ΔsigL, a ˜4-kbfragment, encompassing sigL with its 5′ regulatory region and the distalgenes (MAP4202-MAP4205), was amplified by PCR and cloned into pMV306(24). The M. avium subsp. paratuberculosis ΔsigL strain was transformedwith this recombinant construct and genotype of the complemented strain(M. avium subsp. paratuberculosis ΔsigL::sigL) was identified by PCRanalysis. A similar approach was applied to complement M. tuberculosismutant strain lacking such alternative sigma factor (27).

Stress Phenotype of M. avium Subsp. Paratuberculosis.

M. avium subsp. paratuberculosis cultures were grown to log phase(OD₆₀₀=0.5-1.0) and 200 μl spread on 7H10 plates. For disk diffusionassay (DDA), paper discs (6-mm, Whatman, Piscataway, N.J.) containing 20μl of 0.5, 1, or 1.5M diamide (oxidative stressor) and 1, 2, or 3%sodium dodecyl sulfate (SDS; cell wall stressor) were placed on each ofthe spread plate. Plates were incubated at 37° C. until a thickconfluent lawn developed. To determine sustained effect of stressor onthe viability of bacilli, after washing with PBS, M. avium subsp.paratuberculosis cultures were exposed to the acidified 7H9 broth (pH5.5 obtained by adding HCl) containing 0.3% bovine bile (cell wallstressor) and aliquots were collected at 0, 4, 15, 24, 48, and 72 h tomonitor their viability by colony forming units (CFU) (19).

Cell Culture and Infection.

The mouse macrophage cells (J774A.1) were regularly maintained asdescribed elsewhere (19). To activate macrophages, cells were pretreatedovernight (18 h) with 100 U/ml recombinant murine IFN-γ (Pepro Tech,Rocky Hill, N.J.) before infection with M. avium subsp. paratuberculosisstrains (19). For cell infection studies, wild-type and mutant strainswere added to macrophage monolayers (multiplicity of infection [MOI],20:1). Following incubation at 37° C. in 5% CO₂ for 3 h, macrophagemonolayers were washed twice with warm PBS to remove extracellularbacteria and RPMI-1640 medium containing 5% fetal bovine serum wasadded. Cells were lysed at 1, and 8 days post-infection for bacterialCFU counts. To examine M. avium subsp. paratuberculosis ΔsigL survivalin bovine monocyte-derived macrophages (MDM), MDM cells were isolatedfrom peripheral blood of three cows and cell infection studies wereperformed as described in detail elsewhere (19).

Mouse Infections.

All animal experiments used in this study were performed according tothe protocols approved by the Institutional Animal Care and UseCommittee, UW-Madison. For the virulence study, two groups (N=15 pergroup) of female BALB/c mice (Harlan Laboratories, Indianapolis, USA)were challenged intraperitoneally (i.p) with the wild-type and mutantstrains. Infection inocula (˜2×10⁸ CFU/mouse) of the two strains weresimilar as determined by plate count on the day of infection. Mousegroups (N=5) were sacrificed at 3, 6 and 12 weeks post-infection (WPI),and organ samples were collected for bacterial CFU enumeration andhistopathological examinations as described before (19). For theimmunization studies, female C57BL/6 mice (Taconic, Hudson, N.Y.) wereused. Mock group (N=12) was immunized with PBS buffer while M. aviumsubsp. paratuberculosis ΔsigL mice group (N=14) received ˜2×10⁶ CFU in0.2 ml PBS subcutaneously (s.c.) into the neck scruff twice, 2 weeksapart. Four weeks following booster dose, mice were challenged i.p. with˜7×10⁸ CFU wild-type M. avium subsp. paratuberculosis strain asdetermined by plate count on the day of infection. Mouse groups (N=4-6)were sacrificed at 6 weeks post-immunization and 6 and 12 weekspost-challenge (WPC), and organ samples were collected for bacterial CFUcounts, histopathological examinations, and immune responses.

Evaluation of Immune Responses.

Mouse spleens were collected aseptically and homogenized by gentlemechanical disruption. Following spleen cell isolation, splenocytes werecultured in duplicate in round bottom 96-well tissue culture plates with1×10⁶ cells/well (28). Cells were re-stimulated in vitro with 10 μg/mlJohnin purified protein derivative (PPD) (NVSL, Ames, Iowa) for 48 and72 h. Cell supernatants were collected and analyzed for cytokines byELISA kit according to the manufacturer's instructions (BioLegend, SanDiego, Calif.). To determine humoral immune response, sera were preparedfrom mouse blood and M. avium subsp. paratuberculosis specific antibody(anti-PPDj antibodies) was detected by ELISA using Horseradishperoxidase conjugated rabbit anti-mouse antibody (Pierce, Rockford,Ill.) (29).

Statistical Analysis.

Student's t test was performed to compare differences in mouse immuneresponses and bacterial CFU counts from in vitro stress treatments.Mann-Whitney U test was used to compare bacterial loads in mouse organs.A probability value of <0.05 was considered significant.

Results

Effect of sigL Mutation on Viability of M. avium subsp. paratuberculosisUnder Stress.

Recent analysis of M. avium subsp. paratuberculosis transcriptome duringmacrophage infection suggested that sigL could be an important factorfor M. avium subsp. paratuberculosis survival inside host macrophages(19). To test this hypothesis, we generated a sigL deletion mutant, M.avium subsp. paratuberculosis ΔsigL (FIGS. 4A-B) and examined survivalof this mutant under different stress conditions. Because sigL and itsanti-sigma factor (MAP4202) are likely encoded in an operon (24), weexamined M. avium subsp. paratuberculosis ΔsigL strain for possiblepolarity on the downstream gene, MAP4202. By employingreverse-transcriptase PCR analysis, presence of the MAP4202 transcriptwas confirmed in the ΔsigL mutant (FIG. 4C).

To examine a potential role for sigL in M. avium subsp. paratuberculosisresponse to unfavorable stress conditions, we analyzed the survival ofM. avium subsp. paratuberculosis cultures under both oxidative (diamide)and cell wall stresses (SDS and bovine bile). Growth inhibition zones indisk diffusion assays indicated that M. avium subsp. paratuberculosisΔsigL was susceptible to diamide oxidation. Such phenotypic differencesalso indicated the inability of M. avium subsp. paratuberculosis ΔsigLto survive under SDS stress when compared to the wild-type andcomplemented strains. Bile tolerance was also evaluated by culturing ofthe M. avium subsp. paratuberculosis strains in the presence of 0.3%bovine bile (oxgall). This concentration of bile is likely encounteredby the bacteria within the intestinal content following oral infection(30). In addition, because of the ability of M. avium subsp.paratuberculosis to resist killing by acidic conditions (31), we madeculture broths slightly acidic (pH 5.5) to partially mimic thephysiological condition that M. avium subsp. paratuberculosis wouldencounter following infection in the gastrointestinal tract (e.g.abomasum of a cow and stomach of a human). Survival levels showed asignificant drop in the viability of the M. avium subsp.paratuberculosis ΔsigL at 4 h post-exposure to bovine bile compared tothe wild-type and complemented strains. This difference in bacterialsurvival for M. avium subsp. paratuberculosis ΔsigL was increased bymore than 1.0 log at 24 h and the viability of the mutant continued todecline at later times suggesting that sigL is important in renderingresistance when bacteria experience initial bactericidal barriers in thehost. Because complementation of the M. avium subsp. paratuberculosisΔsigL restored wild-type phenotype under these stress conditions, thecomplemented strain was not included in further experiments.

Intracellular Survival within Macrophages.

Because sigL was up-regulated inside activated murine macrophages (19),we examined intracellular survival of M. avium subsp. paratuberculosisΔsigL in the IFN-γ pretreated murine macrophages. Our analysis showed anincrease in the number of wild-type M. avium subsp. paratuberculosis at8 days relative to the numbers obtained at day 1 post-infection whereasviability of the M. avium subsp. paratuberculosis ΔsigL wassignificantly reduced at this time. To use a more relevant model for M.avium subsp. paratuberculosis infection, we evaluated the persistence ofthe M. avium subsp. paratuberculosis ΔsigL both in resting andIFN-γ-activated bovine monocyte-derived macrophages (MDM cells), thenatural host cell for M. avium subsp. paratuberculosis. At 8 dayspost-infection, the number of wild-type bacilli increased over twofoldcompared to the numbers obtained at day 1 in the resting MDM cells.Specifically, naive and IFN-γ pretreated MDM cells were infected with M.avium subsp. paratuberculosis ΔsigL and wild-type M. avium subsp.paratuberculosis strains. Cells were lysed at 1, and 8 dayspost-infection and numbers of viable bacilli were determined by serialdilutions for CFU plating. The survival level at 8 days was relative tothe viable counts of bacterial strains at day 1. Survival data representthe average of macrophage infections collected from three differentdonor animals with significance levels in Student's t test (*p<0.05).

In contrast, viability of the M. avium subsp. paratuberculosis ΔsigL wassignificantly reduced almost by half indicating a potential function forsigL in defending M. avium subsp. paratuberculosis against intracellularstress. A similar survival trend for the M. avium subsp.paratuberculosis ΔsigL was seen inside IFN-γ pretreated MDM cellswhereas this activation status did not result in more inhibitory effecton the survival of wild-type bacilli. Collectively, survival assaysindicated that deletion of sigL affected M. avium subsp.paratuberculosis viability following exposure to stress conditionssuggesting a significant function for sigL in defending M. avium subsp.paratuberculosis against intracellular insults.

Virulence Analysis of M. avium Subsp. Paratuberculosis ΔsigL Strain.

To evaluate the contribution of SigL to M. avium subsp. paratuberculosisvirulence, we examined persistence of the M. avium subsp.paratuberculosis ΔsigL using the murine model of paratuberculosis. Thesurvival pattern indicated significant attenuation for M. avium subsp.paratuberculosis ΔsigL as early as 3 WPI in all of the examined organs.In the spleen, viability of M. avium subsp. paratuberculosis ΔsigL wasreduced by more than 1-log and 2-log orders relative to the wild-typestrain at 6 and 12 WPI, respectively. Similarly, colonization levels ofthe mutant strain in the liver were significantly lower compared to theparental strain at all examined time points. Interestingly, M. aviumsubsp. paratuberculosis ΔsigL did not persist in the intestines as wewere unable to detect any bacteria (limit of detection 20 CFU) at 6 and12 WPI.

The histological analysis revealed mild to moderate granulomatousinflammation in the liver tissues at both 3 and 6 WPI with either of theM. avium subsp. paratuberculosis strains with higher lymphocyticinfiltration in the mice infected with M. avium subsp. paratuberculosisΔsigL. At 12 WPI, M. avium subsp. paratuberculosis ΔsigL infectedanimals showed less granulomatous inflammation indicating reducedability of M. avium subsp. paratuberculosis ΔsigL compared withwild-type bacilli to establish an infection in animals. In accordancewith the bacterial organ burden data, Ziehl-Neelsen staining showedhigher numbers of acid-fast bacilli in mice liver infected with thewild-type strain relative to the M. avium subsp. paratuberculosis ΔsigLat all examined time points. A similar observation was noticed for thespleen and intestine tissues (data not shown). Both bacterial organcolonization and histological data analyses suggested that M. aviumsubsp. paratuberculosis ΔsigL was attenuated for survival, compared tothe wild-type strain in the mice model of infection.

Immunization with M. avium Subsp. Paratuberculosis ΔsigL.

Because sigL encodes a mycobacterial GGR (32) and was critical for M.avium subsp. paratuberculosis survival in the present study study, weinvestigated the vaccine potential of M. avium subsp. paratuberculosisΔsigL in a challenge model of murine paratuberculosis (FIG. 5A). Toexamine immunogenicity of the mutant strain, groups of mice wereimmunized twice with M. avium subsp. paratuberculosis ΔsigL and 6 weekspost-immunization (WPI) mice organs were analyzed for bacterial content.Two immunizations with this mutant resulted in low colonization (2×10²CFU) in the liver whereas no bacteria were detected (limit of detection20 CFU) in the intestine or spleen (data not shown). To evaluate vaccineinduced immune responses before challenge; ELISA was used to estimatelevels of key cytokines in stimulated spleen cells. Statistical analysisrevealed significantly (p<0.05) high level of IFN-γ secretion in the M.avium subsp. paratuberculosis ΔsigL immunized mice compared to naïveanimals (FIG. 5B). Because of the importance of T-helper 17 cells (33)for intracellular bacterial infection, we examined IL-17A production inthe immunized animals. However, we did not find any detectable levelsfor IL-17A at 6 WPI. Additionally, the mice group vaccinated with M.avium subsp. paratuberculosis ΔsigL had significantly (p<0.05) higheranti-PPDj IgG level (FIG. 5C). Thus, both IFN-γ and IgG data suggestedability of the M. avium subsp. paratuberculosis ΔsigL strain to induceenhanced immune responses.

Protection Against Challenge with M. avium Subsp. Paratuberculosis.

To examine the vaccine potential of sigL-based mutant, groups of micewere vaccinated with PBS (control) or M. avium subsp. paratuberculosisΔsigL live strain and challenged with the virulent M. avium subsp.paratuberculosis K10 strain following 6 WPI (FIG. 5A). At 6 weeks postchallenge (WPC), the ΔsigL mice group had a significant reduction in thebacterial load in spleen and liver (˜0.5 log) compared to thePBS-vaccinated mice (FIG. 6A, B). More importantly, a higher level ofbacterial load reduction (˜1 log) was observed in the intestine (FIG.6C), an important organ for M. avium subsp. paratuberculosis infection.A similar colonization pattern was observed for all examined organs(spleen, liver, intestine) at 12 WPC where the level of bacterialreduction reached >1 log.

For histological examination, we focused our efforts on the liverbecause it is the most reflective organ for M. avium subsp.paratuberculosis infection (34). Liver sections from ΔsigL immunizedanimals displayed lower granulomatous scores and smaller size granulomasthan the PBS-control group at 6 and 12 WPC (FIG. 7A-B). In addition, lownumbers of acid-fast bacilli were observed when liver sections werestained with Ziehl-Neelsen stain, another support for colonization datadiscussed above. Interestingly, sections from the intestines of theΔsigL-immunized mice appeared normal compared to mock infected mice withno detectable acid-fast bacteria in Ziehl-Neelsen stained sections atboth at 6 and 12 WPC (FIG. 7C-D). Overall, reduction in M. avium subsp.paratuberculosis colonization levels combined with histological scoresindicated the ability of the ΔsigL mutant to control tissue damage by achallenge with the virulent strain of M. avium subsp. paratuberculosis.

Expansion of Immune Responses Following Challenge in Immunized Mice.

To evaluate expansion of the cellular immune response followingchallenge, splenocytes of immunized and challenged mice were analyzedfor the production of key cytokines associated with protection againstparatuberculosis (35, 36). As shown in FIG. 6D, PPD-stimulatedsplenocytes from ΔsigL-immunized and challenged mice secretedsignificantly higher levels of IFN-γ than that of control animals at 6WPC, indicating increased levels of T cell activity (T-helper 1 cells)in the animals that received ΔsigL mutant. However, at 12 WPC there wasno significant difference in IFN-γ response between these two groups ofanimals. Our data also showed a better ability, even though notsignificant, of the M. avium subsp. paratuberculosis ΔsigL vaccinatedanimals to induce PPD-specific IL-17A secretion compared to the mockchallenged group at 6 WPC. Taken together, the colonization,histological and immune response levels suggested that M. avium subsp.paratuberculosis ΔsigL induced protective immunity against challengewith virulent M. avium subsp. paratuberculosis strain.

Discussion

Infection with M. avium subsp. paratuberculosis represent a major threat(Johne's disease) to dairy animals (11,14) with the potential spread tohuman with the likely involvement in several diseases (7-10, 12).Earlier reports indicated that M. avium subsp. paratuberculosis count ona large number of sigma factors (N=19) to establish the infection andsurvive diverse stress conditions (19,37). In this study, we havetargeted sigL because of its activation during early macrophageinfection suggesting a role to control important stage(s) of M. aviumsubsp. paratuberculosis pathogenesis following oral infection (19).Moreover, the orthologous sigL deletion mutant, M. tuberculosis ΔsigL,in M. tuberculosis was less lethal for mice relative to the wild-typestrain (32). However, unlike M. tuberculosis ΔsigL (24), our analysisindicated that deletion of sigL affected ability of M. avium subsp.paratuberculosis to survive exposure to intracellular stimuli includingoxidative stress and damaging the mycobacterial cell wall stresses (e.g.diamide and SDS) (38-40). This mutant was also unable to survive bothnaïve and activated macrophages to similar levels, an indication of thesignificant attenuation of this mutant. This intracellular survivaldefect of the mutant was further verified by the poor ability of M.avium subsp. paratuberculosis ΔsigL to replicate in mouse tissues. Bothhistological and bacteriological analyses revealed reduced organcolonization of the M. avium subsp. paratuberculosis ΔsigL with lowinflammatory scores compared to the parental strain. Interestingly, thisresult was in contrast to the reports where orthologous M. tuberculosisΔsigL survived in the mice organs to the same level as the wild-typestrain suggesting a more comprehensive and dynamic role for sigL in M.avium subsp. paratuberculosis survival and pathogenesis (24,32).However, the nature and mechanisms employed by sigL to enable M. aviumsubsp. paratuberculosis virulence remain elusive.

The alternative sigma factor (e.g. sigE) mutant strains were targetedfor the vaccine development and found to provide protection againstinfection with pathogenic bacteria including mycobacteria (41,42). Inour study, observations gained from both in vitro and murine modelexperiments encouraged us to investigate the vaccine potential of M.avium subsp. paratuberculosis ΔsigL as a live attenuated vaccine againstM. avium subsp. paratuberculosis infection in mice. The murine model ofparatuberculosis represent an important screening tool (43,44) toexamine vaccine candidates before testing in a more expensive goatmodel, despite the associated shortcomings of the murine model (nodiarrhea or shedding of bacteria). In our hands, mice that received M.avium subsp. paratuberculosis ΔsigL were very efficient in producingIFN-γ (e.g. 6 WPI), an important cytokine involved in controllingmycobacterial infection (45). The decline in IFN-γ is often associatedwith the onset of clinical JD in ruminants (35,46). Importantly,culturing tissue samples from immunized animals indicated the ability ofM. avium subsp. paratuberculosis ΔsigL to persist in animals followingimmunization but to a low level which could be a critical factor ininducing protective immune responses.

Controlling M. avium subsp. paratuberculosis infection is dependent ondeveloping vaccines that reduce bacterial colonization and shedding frominfected animals. Earlier studies demonstrated the potential use of M.avium subsp. paratuberculosis mutants (e.g. WAg915, M. avium subsp.paratuberculosis ΔleuD) as live attenuated vaccine candidates in themurine model of paratuberculosis (44,47). WAg915 mutant strain (M. aviumsubsp. paratuberculosis ΔppiA), defective in the peptidyl-prolylcis-trans-isomerase, showed mild attenuated phenotype relative to thewild-type strain and provided limited protection in mouse organ only atlater stages after challenge with parental M. avium subsp.paratuberculosis strain (47), whereas the attenuated M. avium subsp.paratuberculosis ΔleuD, defective in leucine biosynthesis, exhibitedsome protection following challenge (44). In our experiment, M. aviumsubsp. paratuberculosis ΔsigL was attenuated in macrophages and in micebut persisted in murine tissues up to the time of challenge. Ability topersist following immunization could be responsible for the generatedprotective immunity, which was more efficient to reduce M. avium subsp.paratuberculosis colonization compared to the Wag915 candidate (47).However, it is not clear whether M. avium subsp. paratuberculosis ΔsigLcould provide better protection compared to the M. avium subsp.paratuberculosis ΔleuD because the previous study examined M. aviumsubsp. paratuberculosis bacilli in mouse tissues by Ziehl-Neelsenstaining which is less sensitive compared to the tissue culturing (48,49). We further evaluated longevity of immune responses in the mousegroups following challenge and these data suggest that M. avium subsp.paratuberculosis ΔsigL immunized mice maintained strong T-cell responseswith secretion of higher IFN-γ and IL-17 at 6 WPC, despite the later notbeing significant compared to the mock infected group.

Overall, an isogenic mutant of M. avium subsp. paratuberculosis lackingsigL had limited ability to survive in macrophages or mice, most likelybecause of a defective bacterial cell wall. Such an attenuated strain ofM. avium subsp. paratuberculosis (ΔsigL) persisted in murine tissuefollowing subcutaneous immunization and generated a robust immuneresponse. The generated immune responses were sufficient to reducetissue colonization and lesion scores in animals following a challengewith the wild-type strain of M. avium subsp. paratuberculosis. Furthervaccine testing in natural hosts of Johne's disease, e.g. goats orcalves, will demonstrate the viability of developing an effectivecontrol strategy against paratuberculosis in both animals and humans. Ingeneral, approaches used to investigate sigL in M. avium subsp.paratuberculosis could be adopted to examine the role and potential useof other global gene regulators in pathogenesis and control ofintracellular pathogens.

Example 3

References cited in this Example are listed in the section of Referencesas “References cited in Example 3.”

Survival of GGR Mutants in the Murine Model of Paratuberculosis.

To assess the role of sigH and sigL in M. ap virulence, we investigatedthe persistence of the each mutant in mice. The initial growth kineticof the wild-type and ΔsigH mutant strains was similar, with an equalburden of bacteria in liver, intestine and spleen up to 6 WPI (FIG. 8).However, at 12 WPI, colonization levels were significantly reduced forΔsigH compared to its parental strain only in spleen and intestine(p<0.01 and p<0.05, respectively), suggesting a role for sigH in thelong term survival of M. ap in mice. For the ΔsigL mutant, bacterialcolonization levels were significantly reduced in spleen, liver andintestine at all times post infection (FIG. 8) compared to K10,suggesting a pivotal role for sigL in the pathogenesis of M. ap.

Interestingly, when the ΔsigH mutant of M. tuberculosis was used tochallenge mice, no differences in bacterial load were observed in mouseorgans compared to the wild-type strain (54), suggesting a moresignificant role for sigH in M. ap. Consistent with the colonizationdata, Ziehl-Neelsen staining of spleen showed higher acid fast bacilliin mice infected with wild-type strain compared to ΔsigH mutant at 12WPI (FIG. 9).

Taken together, our data indicated that both ΔsigH and ΔsigL mutantswere attenuated in the murine model of paratuberculosis compared to thewild-type M. ap strain.

RNA Sequencing for Transcriptional Profiling.

During the last decade, it is evident that the use of DNA microarrayshas greatly contributed to our understanding of the genetic basis ofbacterial infections, including infection with M. ap (42, 55-58).Recently, large scale sequencing approaches were developed for geneexpression profiling to overcome problems associated with DNAmicroarrays (59). The use of Next-generation sequencers enabled us tosequence all transcripts in a given RNA sample, hence named RNA sequence(RNA-Seq) profiling. This naming reflects the ability of the highthroughput sequencers (e.g. HighSeq2000, Roche 454) to be used forin-depth sequencing of millions of transcripts in a single run (59-61).The RNA-Seq approach has already been successfully applied in severalsystems, both mammalian and bacterial, (61) including M. tuberculosisand M. ap in our laboratory (62) (see below). Some key benefits ofRNA-Seq over microarrays include less biased transcriptome dataacquisition, actual sequences for transcripts that can show SNPs andantisense transcription, less complex data analysis; improvedcorrelation between laboratories and increased sensitivity of lowtranscript numbers (63-65).

Because of the global nature of transcriptional regulation under controlof σ factors, we pursued the characterization of the sigH transcriptomeusing RNA-Seq profiling utilizing an Illumina-based sequencer operatedby the University of Wisconsin Biotechnology Center (UWBC). Our stressexperiments showed that the ΔsigH mutant was hypersensitive to elevatedtemperature and diamide exposure, each resulting in impaired growth.Accordingly, we hypothesize that sigH may play an important role indirecting transcriptional control under unfavorable environmentalconditions. To test this hypothesis, both wild-type M. ap K10 and ΔsigHmutant transcriptomes were profiled before and after diamide exposure.Briefly, cultures grown to mid-log phase (OD₆₀₀=0.5) from both K-10 andits isogenic mutant, ΔsigH, were centrifuged (3,200×g) for 15 min at 4°C. for total RNA extraction as outlined before (32). Extracted RNAsamples were treated with DNAse I (Ambion) to eliminate residual genomicDNA and then treated with MICROBExpress (Ambion, Austin) to enrichbacterial mRNA and reduce the amount of rRNA. Following a standardprotocol for cDNA generation in our laboratory (42), all samples weresent to the UWBC for library construction and sequencing on theIllumina/GAIIx sequencing platform using 100-bp, paired end fragments.As a testament of the high resolution analysis of RNA-Seq profiling, wewere not able to detect the presence of only 18 or 20 genes in bothsamples from M. ap K10 or ΔsigH, respectively (Table 2).

TABLE 1 Sequencing run statistics on M. ap samples. M. ap K10 M. ap K10ΔsigH Material Submitted 2 μg mRNA 2 μg mRNA Total Reads 74,747,58253,305,460 Read Length 100 bp 100 bp Total Reads Mapped 57,477,92041,101,069 Reads mapped in pairs 53,359,286 36,887,226 Reads mapped torRNA or tRNA 54,209,373 37,081,401 Reads not mapped 16,903,46411,874,445 Most sequenced mRNA MAP1975 MAP1975 (reads) (1,286,560)(1,390,707) Transcripts not detected     18     20

Examine the Vaccine Potential of GGR M. ap Mutants.

Because they control a large number of genes, we hypothesize that GGRmutants will be attenuated but able generate enough immune responses toserve as live attenuated vaccines that could control JD. To examine thevaccine potential of ΔsigH and ΔsigL mutants as live-attenuatedvaccines, C57BL/6 mice groups (N=11) were vaccinated twice at 2 weeksinterval with 10⁶ CFU/mouse via subcutaneous (S/C.) injection. Thisroute of immunization was chosen after evaluating other routes (oral andintra-peritoneal, I/P.) and was found to yield better immunity (data notshown). In addition, immunized mice via the S/C. showed no M. apcolonization in their organs when examined at 6 weeks post immunization(WPI). At 6 WPI, immunized mice (N=8/group) were challenged with 2×10⁸CFU/mouse of the wild-type strain M. ap K10 via I/P. Mock-immunized micewith (Phosphate Buffered Saline, PBS) showed the highest colonizationlevels of M. ap in their organs following challenge with M. ap K10. Onthe other hand, mice vaccinated with the attenuated mutants (ΔsigH andΔsigL) showed a significant reduction in bacterial colony levels in theintestine at 6 weeks post challenge (WPC) (FIG. 10). OnlyΔsigL-vaccinated group showed significant reduction in both liver andspleen at this time point. By 12 WPC, only ΔsigL-vaccinated group showedmore than 1 log reduction in intestine in all organs (intestine, liver,spleen) compared to the control group (FIG. 10). Overall, ΔsigLimmunization was more effective in inducing protection against virulentM. ap K10 strain compared to ΔsigH vaccination.

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I claim:
 1. A mycobacterium mutant, wherein the mycobacterium isselected from the group consisting of Mycobacterium avium subspeciesparatuberculosis (M. ap) and Mycobacterium bovis (M. bovis), comprisingat least one mutation in at least one gene sequence selected from thegroup consisting of sigL and ECF-1, which encode a global gene regulator(GGR), wherein the GGR gene is at least partially inactivated.
 2. Themutant of claim 1, wherein the mycobacterium is M. ap.
 3. The mutant ofclaim 1, wherein the mycobacterium is M. bovis.
 4. A mycobacteriummutant, wherein the mycobacterium is Mycobacterium avium subspeciesparatuberculosis (M. ap), comprising at least one mutation in sigL (SEQID NO:2), which encodes a global gene regulator (GGR), or a sequencesubstantially identical to SEQ ID NO:2, wherein the GGR is at leastpartially inactivated.
 5. The mutant of claim 1, wherein the GGR is M.ap ECF-1 (SEQ ID NO:4) or a sequence substantially identical to SEQ IDNO:4.
 6. The mutant of claim 1, wherein the inactivation is achieved byat least partial deletion of a gene sequence encoding a GGR.
 7. Themutant of claim 6, wherein the inactivation is achieved by a completedeletion of a gene sequence encoding a GGR.
 8. The mutant of claim 1,wherein the inactivation is achieved by at least partial transposoninsertion of a gene sequence encoding a GGR.
 9. The mutant of claim 8,wherein the inactivation is achieved by a complete transposon insertionof a gene sequence encoding a GGR.
 10. The mutant of claim 1, whereinthe inactivation is achieved using a partial anti-sense construct of agene sequence encoding a GGR.
 11. The mutant of claim 10, wherein theinactivation is achieved using a complete anti-sense construct of a genesequence encoding a GGR.
 12. An isolated mycobacterial organismcomprising a mutation according to claim 1, wherein the mycobacterium isselected from the group consisting of Mycobacterium avium subspeciesparatuberculosis (M. ap) and Mycobacterium bovis (M. bovis).
 13. Amycobacterium vaccine comprising a mycobacterium mutant according toclaim
 1. 14. A mycobacterium vaccine comprising an isolatedmycobacterial organism comprising a mycobacterium mutant, wherein themycobacterium is selected from the group consisting of Mycobacteriumavium subspecies paratuberculosis (M. ap) and Mycobacterium bovis (M.bovis), comprising at least one mutation in sigL, which encodes a globalgene regulator (GGR), wherein the GGR gene is at least partiallyinactivated.
 15. The vaccine of claim 14, wherein the vaccine isphysically inactivated.
 16. The vaccine of claim 15, wherein the vaccineis heat inactivated.
 17. The vaccine of claim 14, wherein the vaccine ischemically inactivated.
 18. The vaccine of claim 17, wherein the vaccineis formaldehyde inactivated.
 19. The vaccine of claim 14, wherein thevaccine is a live attenuated vaccine.